Novel pd-l1 binding polypeptides for imaging

ABSTRACT

Provided herein are novel  10 Fn3 domains which specifically bind to PD-L1, as well as imaging agents based on the same for diagnostics.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/084,298, entitled “Novel PD-L1 Binding Polypeptides for Imaging”filed Nov. 25, 2014, the contents of which are hereby incorporated byreference.

BACKGROUND

Programmed Death Ligand-1 (PD-L1) is a surface glycoprotein ligand forPD-1, a key immune checkpoint receptor expressed by activated T and Bcells and mediates immunosuppression, which is found on bothantigen-presenting cells and human cancers and downregulaes T cellactivation and cytokine secretion by binding to PD-1 (Freeman et al.,2000; Latchman et al, 2001). Inhibition of the PD-L1/PD-1 interactionallows for potent anti-tumor activity in preclinical models, andantibodies that disrupt this interaction have entered clinical trialsfor the treatment of cancer (U.S. Pat. Nos. 8,008,449 and 7,943,743;Brahmer et al., 2010; Topalian et al., 2012b; Brahmer et al, 2012; Flieset al., 2011; Pardoll, 2012; Hamid and Carvajal, 2013).

PET, or Positron Emission Tomography, is a non-invasive, nuclearmedicine technique that produces a three-dimensional image of variousmolecular processes within the body, or the location of proteinsassociated with disease pathology. The methodology detects pairs ofgamma rays emitted indirectly by a positron-emitting radionuclide(tracer) introduced into the body on a biologically active molecule. PETimaging tools have a wide variety of uses for drug development and havea unique translational medicine advantage, in that the same tool couldbe used both preclinically and clinically. Examples include directvisualization of in vivo saturation of targets; monitoring uptake innormal tissues to anticipate toxicity or patient to patient variation;quantifying diseased tissue; tumor metastasis; monitoring drug efficacyover time, or resistance over time, and more.

Described herein are novel anti-PD-L1 Adnectins suitable for use asdiagnostic/imaging agents, for example, for use in positron emissiontomography.

SUMMARY

The present invention is based, at least in part, on the discovery ofnew anti-human PD-L1 Adnectins which are useful as diagnostic/imagingagents, for example, for use in positron emission tomography. Theseagents are useful in, e.g., the differentiation of PD-L1 expressingcells from non-PD-L1 expressing cells, e.g., tumor cells, and thedifferentiation of PD-L1 expressing tissue from non-PD-L1 expressingtissue, e.g., cancer tissue.

In one aspect, provided herein is a polypeptide comprising a fibronectintype III tenth domain (¹⁰Fn3), wherein (a) the ¹⁰Fn3 domain comprisesAB, BC, CD, DE, EF, and FG loops, (b) the ¹⁰Fn3 has at least one loopselected from loop BC, DE, and FG with an altered amino acid sequencerelative to the sequence of the corresponding loop of the human ¹⁰Fn3domain (SEQ ID NO: 1), and (c) the polypeptide specifically binds toPD-L1. In certain embodiments, the polypeptide binds to PD-L1 with aK_(D) of 500 mM or less, for example, 100 mM or less.

In certain embodiments, the ¹⁰Fn3 domain comprises BC, DE, and FG loopscomprising the amino acid sequences of:

(1) SEQ ID NOs: 6, 7, and 8, respectively;

(2) SEQ ID NOs: 21, 22, and 23, respectively;

(3) SEQ ID NOs: 36, 37, and 38, respectively;

(4) SEQ ID NOs: 51, 52, and 53, respectively;

(5) SEQ ID NOs: 66, 67, and 68, respectively;

(6) SEQ ID NOs: 81, 82, and 83, respectively;

(7) SEQ ID NOs: 97, 98, and 99, respectively;

(8) SEQ ID NOs: 113, 114, and 115, respectively;

(9) SEQ ID NOs: 124, 125 and 126, respectively;

(10) SEQ ID NOs: 135, 136 and 137, respectively;

(11) SEQ ID NOs: 146, 147 and 148, respectively;

(12) SEQ ID NOs: 157, 158 and 159, respectively;

(13) SEQ ID NOs: 168, 169 and 170, respectively;

(14) SEQ ID NOs: 179, 180 and 181, respectively;

(15) SEQ ID NOs: 190, 191 and 192, respectively;

(16) SEQ ID NOs: 201, 202 and 203, respectively;

(17) SEQ ID NOs: 212, 213 and 214, respectively;

(18) SEQ ID NOs: 223, 224 and 225, respectively;

(19) SEQ ID NOs: 234, 235, and 236, respectively;

(20) SEQ ID NOs: 245, 246 and 247, respectively;

(21) SEQ ID NOs: 256, 257 and 258, respectively;

(22) SEQ ID NOs: 267, 268 and 269, respectively;

(23) SEQ ID NOs: 278, 279 and 280, respectively;

(24) SEQ ID NOs: 289, 290 and 291, respectively;

(25) SEQ ID NOs: 300, 301 and 302, respectively;

(26) SEQ ID NOs: 311, 312 and 313, respectively;

(27) SEQ ID NOs: 322, 323 and 324, respectively;

(28) SEQ ID NOs: 333, 334 and 335, respectively;

(29) SEQ ID NOs: 344, 345 and 346, respectively;

(30) SEQ ID NOs: 355, 356 and 357, respectively;

(31) SEQ ID NOs: 366, 367 and 368, respectively;

(32) SEQ ID NOs: 377, 378 and 379, respectively;

(33) SEQ ID NOs: 388, 389 and 390 respectively;

(34) SEQ ID NOs: 399, 400 and 401, respectively;

(35) SEQ ID NOs: 410, 411 and 412, respectively;

(36) SEQ ID NOs: 421, 422 and 423, respectively;

(37) SEQ ID NOs: 432, 433 and 434 respectively;

(38) SEQ ID NOs: 443, 444 and 445, respectively;

(39) SEQ ID NOs: 454, 455 and 456, respectively;

(40) SEQ ID NOs: 465, 466 and 467, respectively;

(41) SEQ ID NOs: 476, 477 and 478, respectively;

(42) SEQ ID NOs: 487, 488 and 489, respectively;

(43) SEQ ID NOs: 498, 499 and 500, respectively;

(44) SEQ ID NOs: 509, 510 and 511, respectively;

(45) SEQ ID NOs: 520, 521 and 522, respectively;

(46) SEQ ID NOs: 531, 530 and 531, respectively;

(47) SEQ ID NOs: 542, 543 and 544, respectively;

(48) SEQ ID NOs: 553, 554 and 555, respectively; or

(49) SEQ ID NOs: 564, 565 and 566, respectively.

In certain embodiments, the polypeptide comprises an amino acid sequenceat least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a sequenceset forth in Table 3, e.g, any one of SEQ ID NO: 5, 20, 35, 50, 65, 80,96, 112, 123, 134, 145, 156, 167, 178, 189, 200, 211, 222, 233, 244,255, 266, 277, 288, 299, 310, 321, 332, 343, 354, 365, 376, 387, 398,409, 420, 431, 442, 453, 464, 475, 486, 497, 508, 519, 530, 541, 552 and563. In certain embodiments, the polypeptide comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to thenon-BC, DE, and FG loop regions of SEQ ID NO: 5, 20, 35, 50, 65, 80, 96,112, 123, 134, 145, 156, 167, 178, 189, 200, 211, 222, 233, 244, 255,266, 277, 288, 299, 310, 321, 332, 343, 354, 365, 376, 387, 398, 409,420, 431, 442, 453, 464, 475, 486, 497, 508, 519, 530, 541, 552 or 563.In certain embodiments, the polypeptide comprises an amino acid sequenceat least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to an amino acidsequence selected from the group consisting of: SEQ ID NOs: 9-15, 24-30,39-45, 54-60, 69-75, 84-91, 100-107, 116-122, 127-133, 138-144, 150-155,160-166, 171-177, 182-188, 193-199, 204-210, 215-221, 227-232, 237-243,248-254, 259-265, 271-276, 291-287, 292-298, 303-309, 314-320, 325-331,337-342, 347-353, 358-364, 369-375, 380-386, 391-397, 402-408, 413-419,424-430, 435-441, 446-452, 457-463, 468-474, 479-485, 490-496, 501-507,512-518, 523-529, 534-540, 545-551, and 556-562. In certain embodiments,the polypeptide comprises an amino acid sequence at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop regionsof an amino acid sequence selected from the group consisting of: SEQ IDNOs: 9-15, 24-30, 39-45, 54-60, 69-75, 84-91, 100-107, 116-122, 127-133,138-144, 150-155, 160-166, 171-177, 182-188, 193-199, 204-210, 215-221,227-232, 237-243, 248-254, 259-265, 271-276, 291-287, 292-298, 303-309,314-320, 325-331, 337-342, 347-353, 358-364, 369-375, 380-386, 391-397,402-408, 413-419, 424-430, 435-441, 446-452, 457-463, 468-474, 479-485,490-496, 501-507, 512-518, 523-529, 534-540, 545-551, and 556-562.

In certain embodiments, the polypeptide comprises an N-terminal leaderselected from the group consisting of SEQ ID NOs: 574-583, and/or aC-terminal tail selected from the group consisting of SEQ ID NOs:584-618 or PmCn, wherein P is proline, and wherein m is an integer thatis at least 0 (e.g., 0, 1 or 2) and n is an integer of at least 1 (e.g.,1 or 2).

In certain embodiments, the polypeptide comprises one or morepharmacokinetic (PK) moieties selected from the group consisting ofpolyethylene glycol, sialic acid, Fc, Fc fragment, transferrin, serumalbumin, a serum albumin binding protein, and a serum immunoglobulinbinding protein. In certain embodiments, the PK moiety and thepolypeptide are linked via at least one disulfide bond, a peptide bond,a polypeptide, a polymeric sugar or a polyethylene glycol moiety. Incertain embodiments, the PK moiety and the polypeptide are linked via alinker with an amino acid sequence selected from the group consisting ofSEQ ID NOs: 629-678.

Provided herein are nucleic acids encoding the polypeptides, as well asvectors and cells comprising the nucleic acids, described herein. Incertain embodiments, the nucleic acid comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 16-19, 31-34, 46-49,61-64, 76-79, 92-95, and 108-111.

Provided herein are compositions comprising the polypeptides describedherein, and a carrier. For example, the compositions described hereincomprise a polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 5, 9-15, 20, 24-30, 35, 39-45, 50,54-60, 65, 69-75, 80, 84-91, 96, 100-107, 112, 116-122, 123, 127-133,134, 138-144, 145, 150-155, 156, 160-166, 167, 171-177, 178, 182-188,189, 193-199, 200, 204-210, 211, 215-221, 222, 227-232, 233, 237-243,244, 248-254, 255, 259-265, 266, 271-276, 277, 291-287, 288, 292-298,299, 303-309, 310, 314-320, 321, 325-331, 332, 337-342, 343, 347-353,354, 358-364, 365, 369-375, 376, 380-386, 387, 391-397, 398, 402-408,409, 413-419, 420, 424-430, 431, 435-441, 442, 446-452, 453, 457-463,464, 468-474, 475, 479-485, 486, 490-496, 497, 501-507, 508, 512-518,519, 523-529, 530, 534-540, 541, 545-551, 552, and 556-562, and acarrier.

Provided herein are imaging agents comprising the polypeptide disclosedherein. In certain embodiments, the imaging agent comprises apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 9-15, 20, 24-30, 35, 39-45, 50, 54-60, 65,69-75, 80, 84-91, 96, 100-107, 112, 116-122, 123, 127-133, 134, 138-144,145, 150-155, 156, 160-166, 167, 171-177, 178, 182-188, 189, 193-199,200, 204-210, 211, 215-221, 222, 227-232, 233, 237-243, 244, 248-254,255, 259-265, 266, 271-276, 277, 291-287, 288, 292-298, 299, 303-309,310, 314-320, 321, 325-331, 332, 337-342, 343, 347-353, 354, 358-364,365, 369-375, 376, 380-386, 387, 391-397, 398, 402-408, 409, 413-419,420, 424-430, 431, 435-441, 442, 446-452, 453, 457-463, 464, 468-474,475, 479-485, 486, 490-496, 497, 501-507, 508, 512-518, 519, 523-529,530, 534-540, 541, 545-551, 552, and 556-562.

In certain embodiments, the imaging agent comprises a detectable label.In certain embodiments, the imaging agent comprises a polypeptidedisclosed herein, a chelating agent, and a detectable label. In certainembodiments, the imaging agent comprises a polypeptide disclosed herein,a bifunctional chelator or conjugating (BFC) moiety and a detectablelabel. In certain embodiments, the detectable label is a prostheticgroup containing a radionuclide. In certain embodiments, the detectablelabel is detectable by positron emission tomography.

In certain embodiments, the chelating agent and/or bifunctional chelatoror conjugating (BFC) moiety is selected from the group consisting ofDFO, DOTA, CB-DO2A, 3p-C-DEPA, TCMC, DBCO, DIBO, BARAC, DIMAC, Oxo-DO3A,TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, diamsar, NODASA,NODAGA, NOTA, NETA, TACN-TM, DTPA, 1B4M-DTPA, CHX-A″-DTPA, TRAP, NOPO,AAZTA, DATA, H₂dedpa, H₄octapa, H₂azapa, H₅decapa, H₆phospa, HBED,SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA.

In certain embodiments, the detectable label is a radionuclide, forexample,

⁶⁴Cu, ¹²⁴I, ^(76/77)Br, ⁸⁶Y, ⁸⁹Zr, ⁶⁸Ga, ¹⁸F, ¹¹C, ¹²⁵I, ¹²⁴I, ¹³¹I,¹²³I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁶⁸Ga, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br,⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, or ¹⁵³Sm.

In certain embodiments, the chelating agent is NODAGA and theradionuclide is ⁶⁴Cu. In certain embodiments, the imaging agentcomprises an anti-PD-L1 polypeptide (e.g., an anti-PD-L1 Adnectindescribed herein, e.g., an anti-PD-L1 Adnectin comprising the amino acidsequence set forth in SEQ ID NO: 80 or 96), the chelating agent NODAGA,and the radionuclide ⁶⁴Cu.

In certain embodiments, the imaging agent comprises an anti-PD-L1polypeptide (e.g., an anti-PD-L1 Adnectin described herein, e.g., ananti-PD-L1 Adnectin comprising the amino acid sequence set forth in SEQID NO: 80 or 96), a bifunctional chelator or conjugating (BFC) moiety,and a prosthetic group comprising the radionuclide ¹⁸F. In certainembodiments, the imaging agent has the following structure:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the imaging agent has the following structure:

In some embodiments, the BFC is a cyclooctyne comprising a reactivegroup that forms a covalent bond with an amine, carboxyl, carbonyl orthiol functional group on the protein. In some embodiments, thecyclooctyne is selected from the group consisting of dibenzocyclooctyne(DIBO), biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC)and dibenzocyclooctyne (DBCO). In some embodiments, the cyclooctyne isDBCO.

In some embodiments, the BFC is DBCO-PEG4-NHS-Ester,DBCO-Sulfo-NHS-Ester, DBCO-PEG4-Acid, DBCO-PEG4-Amine orDBCO-PEG4-Maleimide. In some embodiments, the BFC isDBCO-PEG4-Maleimide.

In certain embodiments, the imaging agent has the structure:

wherein X is a polypeptide comprising the amino acid sequence of any oneof SEQ ID NOs: 13, 28, 43, 58, 73, 88, 104, 120, 131, 142, 153, 164,175, 186, 197, 208, 219, 230, 241, 252, 263, 274, 285, 296, 307, 318,329, 340, 351, 362, 373, 384, 395, 406, 417, 428, 439, 450, 461, 472,483, 494, 505, 516, 527, 538, 549, 560 and 571. In certain embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 88. In certain embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 104.

Provided herein are kits comprising an anti-PD-L1 Adnectin compositionand/or imaging agent described herein, and instructions for use.

Provided herein is a method of detecting PD-L1 in a sample, the methodcomprising contacting the sample with an anti-PD-L1 Adnectin, anddetecting PD-L1.

Provided herein is a method of detecting PD-L1 positive cells in asubject comprising administering to the subject an imaging agentcomprising an anti-PD-L1 Adnectin, and detecting the imaging agent, thedetected imaging agent defining the location of the PD-L1 positive cellsin the subject.

Provided herein is a method of detecting PD-L1-expressing tumors in asubject comprising administering to the subject an imaging agentcomprising an anti-PD-L1 Adnectin, and detecting the imaging agent, thedetected imaging agent defining the location of the tumor in thesubject. In certain embodiments, the imaging agent is detected bypositron emission tomography.

Provided herein is a method of obtaining an image of an imaging agentcomprising an anti-PD-L1 Adnectin, the method comprising,

-   -   a) administering the imaging agent to a subject; and    -   b) imaging in vivo the distribution of the imaging agent by        positron emission tomography.

Provided herein is a method of obtaining a quantitative image of tissuesor cells expressing PD-L1, the method comprising contacting the cells ortissue with an imaging agent comprising an anti-PD-L1 Adnectin, anddetecting or quantifying the tissue expressing PD-L1 using positronemission tomography.

Provided herein is a method for detecting a PD-L1-expressing tumorcomprising administering an imaging-effective amount of an imaging agentcomprising an anti-PD-L1 Adnectin to a subject having a PD-L1-expressingtumor, and detecting the radioactive emissions of said imaging agent inthe tumor using positron emission tomography, wherein the radioactiveemissions are detected in the tumor.

Provided herein is a method of diagnosing the presence of aPD-L1-expressing tumor in a subject, the method comprising

(a) administering to a subject in need thereof an imaging agentcomprising an anti-PD-L1 Adnectin; and

(b) obtaining an radio-image of at least a portion of the subject todetect the presence or absence of the imaging agent;

wherein the presence and location of the imaging agent above backgroundis indicative of the presence and location of the disease.

Provided herein is a method of monitoring the progress of an anti-tumortherapy against PD-L1-expressing tumors in a subject, the methodcomprising

(a) administering to a subject in need thereof an imaging agentcomprising an anti-PD-L1 Adnectin at a first time point and obtaining animage of at least a portion of the subject to determine the size of thetumor;(b) administering an anti-tumor therapy to the subject;(c) administering to the subject the imaging agent at one or moresubsequent time points and obtaining an image of at least a portion ofthe subject at each time point; wherein the dimension and location ofthe tumor at each time point is indicative of the progress of thedisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the core amino acid sequences of exemplaryanti-PD-L1 Adnectins described herein. The BC, DE, and FG loops areunderlined. Wildtype (WT) (SEQ ID NO: 2), ATI-968 (SEQ ID NO: 5),ATI-964 (SEQ ID NO: 20), ATI-967 (SEQ ID NO: 65), A02 (SEQ ID NO: 80),E01 (SEQ ID NO: 96), ATI-965 (SEQ ID NO: 35), and ATI-966 (SEQ ID NO:50).

FIG. 2 is a schematic for the chemical synthesis of[¹⁸F]-E01-4PEG-DBCO-FPPEGA. The E01 portion of the molecule has thesequence set forth in SEQ ID NO: 104.

FIG. 3 is a graph demonstrating discrimination of hPD-L1-positive L2987cells from hPD-L1-negative HT-29 cells with the ⁶⁴Cu-E01 anti-PD-L1Adnectin (with a NODAGA chelator). Specificity was confirmed by thereduction of cell-associated ⁶⁴Cu-E01 when co-incubated with excess cold(unlabeled) E01 Adnectin.

FIG. 4A is a PET image depicting the discrimination of hPD-L1 (+) fromhPD-L1 (−) tumors in bilateral xenograft mice with a NODAGA-⁶⁴Cu-labeledA02 anti-PD-L1 Adnectin. Shown are summed 0 to 2 hour images showingareas of probe residence. Bright areas are tissues of greatest occupancyduring exposure.

FIG. 4B is a graph depicting a time course of tumor labeling in hPD-L1(+) [L2987] tumors. hPD-L1(−) [HT29] tumors and pulse chase experimentin hPD-L1(+) systems [L2987 block] show the specificity of thelabelling.

FIG. 5 is a graph depicting tissue distribution of the ¹⁸F-labeled A02anti-PD-L1 Adnectin radiotracer in mice bearing bilateral hPD-L1(+)L2987 and hPD-L1(−) HT-29 xenografts as measured ex vivo by gammacounter.

FIG. 6 is a composite image of ¹⁸F-labeled E01 anti-PD-L1 Adnectindistribution in cynomolgus monkey.

FIG. 7 is an image depicting in vitro autoradiography of xenograft andhuman lung tissues labelled with the 0.25 nM ¹⁸F-DBCO-A02 anti-PD-L1Adnectin co-incubated with the indicated concentrations of cold A02Adnectin.

FIG. 8 depicts immunohistochemistry images of xenograft and human lungtumor specimens labelled with anti-PD-L1 Adnectins to demonstrate tumorexpression of hPD-L1.

FIG. 9 shows a reaction scheme for synthesizing[¹⁸F]-A02-4PEG-DBCO-FPPEGA. The same reaction scheme was used to labelthe E01 adnectin.

FIG. 10 is a schematic of the GE TRACERlab FX2 N Synthesis module forautomated synthesis of [¹⁸F]-FPPEGA.

FIG. 11 is a schematic of the Synthera Synthesis module (IBA) forautomated synthesis of [¹⁸F]-FPPEGA.

FIG. 12 depicts exemplary competition curves of anti-PD-L1 Adnectins.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by the skilled artisan.Although any methods and compositions similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention, the preferred methods and compositions are described herein.

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulateT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7. PD-L1 is also referred to asCD274, B7-H, B7H1, PDCD1L1, and PDCD1LG1.

“Polypeptide” as used herein refers to any sequence of two or more aminoacids, regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D). Thepeptides described herein are proteins derived from the tenth type IIIdomain of fibronectin that have been modified to bind specifically toPD-L1 and are referred to herein as, “anti-PD-L1 Adnectin” or “PD-L1Adnectin.”

A “polypeptide chain,” as used herein, refers to a polypeptide whereineach of the domains thereof is joined to other domain(s) by peptidebond(s), as opposed to non-covalent interactions or disulfide bonds.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing condition using Coomassie blue or, preferably, silver stain.Isolated polypeptide includes the polypeptide in situ within recombinantcells since at least one component of the polypeptide's naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

A “region” of a ¹⁰Fn3 domain as used herein refers to either a loop (AB,BC, CD, DE, EF and FG), a β-strand (A, B, C, D, E, F and G), theN-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO: 1),or the C-terminus (corresponding to amino acid residues 93-94 of SEQ IDNO: 1) of the human ¹⁰Fn3 domain.

A “scaffold region” refers to any non-loop region of a human ¹⁰Fn3domain. The scaffold region includes the A, B, C, D, E, F and Gβ-strands as well as the N-terminal region (amino acids corresponding toresidues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acidscorresponding to residues 93-94 of SEQ ID NO: 1 and optionallycomprising the 7 amino acids constituting the natural linker between the10^(th) and the 11^(th) repeat of the Fn3 domain in human fibronectin).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR™) software. Those skilledin the art can readily determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared. For example, the %amino acid sequence identity of a given amino acid sequence A to, with,or against a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows: 100 times the fraction X/Y where Xis the number of amino acid residues scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of A andB, and where Y is the total number of amino acid residues in B. It willbe appreciated that where the length of amino acid sequence A is notequal to the length of amino acid sequence B, the % amino acid sequenceidentity of A to B will not equal the % amino acid sequence identity ofB to A.

As used herein, the term “Adnectin binding site” refers to the site orportion of a protein (e.g., PD-L1) that interacts or binds to aparticular Adnectin. Adnectin binding sites can be formed fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Adnectin binding sites formed bycontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas Adnectin binding sites formed by tertiary folding aretypically lost on treatment of denaturing solvents.

An Adnectin binding site for an anti-PD-L1 Adnectin described herein maybe determined by application of standard techniques typically used forepitope mapping of antibodies including, but not limited to proteasemapping and mutational analysis. Alternatively, an Adnectin binding sitecan be determined by competition assay using a reference Adnectin orantibody which binds to the same polypeptide, e.g., PD-L1 (as furtherdescribed infra in the section “Cross-Competing Adnectins and/orAdnectins that Bind to the Same Adnectin Binding Site.” If the testAdnectin and reference molecule (e.g., another Adnectin or antibody)compete, then they bind to the same Adnectin binding site or to Adnectinbinding sites sufficiently proximal such that binding of one moleculeinterferes with the other.

The terms “specifically binds,” “specific binding,” “selective binding,and “selectively binds,” as used interchangeably herein in the contextof Adnectins binding to PD-L1 refers to an Adnectin that exhibitsaffinity for PD-L1, but does not significantly bind (e.g., less thanabout 10% binding) to a different polypeptides as measured by atechnique available in the art such as, but not limited to, Scatchardanalysis and/or competitive binding assays (e.g., competition ELISA,BIACORE assay). The term is also applicable where e.g., a binding domainof an Adnectin described herein is specific for PD-L1.

The term “preferentially binds” as used herein in the context ofAdnectins binding to PD-L1 refers to the situation in which an Adnectindescribed herein binds PD-L1 at least about 20% greater than it binds adifferent polypeptide as measured by a technique available in the artsuch as, but not limited to, Scatchard analysis and/or competitivebinding assays (e.g., competition ELISA, BIACORE assay).

As used herein in the context of Adnectins, the term “cross-reactivity”refers to an Adnectin which binds to more than one distinct proteinhaving identical or very similar Adnectin binding sites.

The term “K_(D)” as used herein in the context of Adnectins binding toPD-L1, is intended to refer to the dissociation equilibrium constant ofa particular Adnectin-protein (e.g., PD-L1) interaction or the affinityof an Adnectin for a protein (e.g., PD-L1), as measured using a surfaceplasmon resonance assay or a cell binding assay. A “desired K_(D),” asused herein, refers to a K_(D) of an Adnectin that is sufficient for thepurposes contemplated. For example, a desired K_(D) may refer to theK_(D) of an Adnectin required to elicit a functional effect in an invitro assay, e.g., a cell-based luciferase assay.

The term “k_(a)”, as used herein in the context of Adnectins binding toa protein, is intended to refer to the association rate constant for theassociation of an Adnectin into the Adnectin/protein complex.

The term “k_(d)”, as used herein in the context of Adnectins binding toa protein, is intended to refer to the dissociation rate constant forthe dissociation of an Adnectin from the Adnectin/protein complex.

The term “IC₅₀”, as used herein in the context of Adnectins, refers tothe concentration of an Adnectin that inhibits a response, either in anin vitro or an in vivo assay, to a level that is 50% of the maximalinhibitory response, i.e., halfway between the maximal inhibitoryresponse and the untreated response.

The term “PK” is an acronym for “pharmacokinetic” and encompassesproperties of a compound including, by way of example, absorption,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” as used herein refers to any protein, peptide,or moiety that affects the pharmacokinetic properties of a biologicallyactive molecule when fused to or administered together with thebiologically active molecule. Examples of a PK modulation protein or PKmoiety include PEG, human serum albumin (HSA) binders (as disclosed inU.S. Publication Nos. 2005/0287153 and 2007/0003549, PCT PublicationNos. WO 2009/083804 and WO 2009/133208), human serum albumin andvariants thereof, transferrin and variants thereof, Fc or Fc fragmentsand variants thereof, and sugars (e.g., sialic acid).

The “serum half-life” of a protein or compound is the time taken for theserum concentration of the polypeptide to be reduced by 50%, in vivo,for example due to degradation of the sequence or compound and/orclearance or sequestration of the sequence or compound by naturalmechanisms. The half-life can be determined in any manner known per se,such as by pharmacokinetic analysis. Suitable techniques will be clearto the person skilled in the art, and may for example generally involvethe steps of suitably administering to a subject a suitable dose of theamino acid sequence or compound described herein; collecting bloodsamples or other samples from the subject at regular intervals;determining the level or concentration of the amino acid sequence orcompound described herein in said blood sample; and calculating, from (aplot of) the data thus obtained, the time until the level orconcentration of the amino acid sequence or compound described hereinhas been reduced by 50% compared to the initial level upon dosing.Reference is, for example, made to the standard handbooks, such asKenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al., Pharmacokinete Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

Half-life can be expressed using parameters such as the t_(1/2)-alpha,t_(1/2)-beta, HL_Lambda_z, and the area under the curve (AUC). In thepresent specification, an “increase in half-life” refers to an increasein any one of these parameters, any two of these parameters, any threeof these parameters or all four of these parameters. An “increase inhalf-life” in particular refers to an increase in the t_(1/2)-beta,and/or HL_Lambda_z, either with or without an increase in thet_(1/2)-alpha and/or the AUC or both.

The term “detectable” refers to the ability to detect a signal over thebackground signal. The term “detectable signal” is a signal derived fromnon-invasive imaging techniques such as, but not limited to, positronemission tomography (PET). The detectable signal is detectable anddistinguishable from other background signals that may be generated fromthe subject. In other words, there is a measurable and statisticallysignificant difference (e.g., a statistically significant difference isenough of a difference to distinguish among the detectable signal andthe background, such as about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%,or 40% or more difference between the detectable signal and thebackground) between the detectable signal and the background. Standardsand/or calibration curves can be used to determine the relativeintensity of the detectable signal and/or the background.

A “detectably effective amount” of a composition comprising an imagingagent described herein is defined as an amount sufficient to yield anacceptable image using equipment that is available for clinical use. Adetectably effective amount of an imaging agent provided herein may beadministered in more than one injection. The detectably effective amountcan vary according to factors such as the degree of susceptibility ofthe individual, the age, sex, and weight of the individual,idiosyncratic responses of the individual, and the like. Detectablyeffective amounts of imaging compositions can also vary according toinstrument and methodologies used. Optimization of such factors is wellwithin the level of skill in the art. In certain embodiments, a PD-L1imaging agent, e.g., those described herein, provides a differentiationfactor (i.e., specific signal to background signal) of 2 or more, e.g.,3, 4, 5 or more.

The term “bioorthogonal chemistry” refers to any chemical reaction thatcan occur inside of living systems without interfering with nativebiochemical processes. The term includes chemical reactions that arechemical reactions that occur in vitro at physiological pH in, or in thepresence of water. To be considered bioorthogonol, the reactions areselective and avoid side-reactions with other functional groups found inthe starting compounds. In addition, the resulting covalent bond betweenthe reaction partners should be strong and chemically inert tobiological reactions and should not affect the biological activity ofthe desired molecule.

The term “click chemistry” refers to a set of reliable and selectivebioorthogonal reactions for the rapid synthesis of new compounds andcombinatorial libraries. Properties of for click reactions includemodularity, wideness in scope, high yielding, stereospecificity andsimple product isolation (separation from inert by-products bynon-chromatographic methods) to produce compounds that are stable underphysiological conditions. In radiochemistry and radiopharmacy, clickchemistry is a generic term for a set of labeling reactions which makeuse of selective and modular building blocks and enable chemoselectiveligations to radiolabel biologically relevant compounds in the absenceof catalysts. A “click reaction” can be with copper, or it can be acopper-free click reaction.

The term “prosthetic group” or “bifunctional labeling agent” refers to asmall organic molecule containing a radionulide (e.g., ¹⁸F) that iscapable of being linked to peptides or proteins.

The term “chelator ligand” as used herein with respect toradiopharmaceutical chemistry refers to a bifunctional chelator orconjugating (BFC) moiety, which are used interchangeably herein, thatcovalently links a radiolabeled prosthetic group to a biologicallyactive targeting molecule (e.g., peptide or protein). BFCs utilizefunctional groups such as carboxylic acids or activated esters for amidecouplings, isothiocyanates for thiourea couplings and maleimides forthiol couplings.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to an animal, preferably a mammal (including a nonprimateand a primate), e.g., a human. In certain embodiments, a subject has adisease or disorder or condition that would benefit from a decreasedlevel or decreased bioactivity of PD-L1. In certain embodiments, asubject is at risk of developing a disorder, disease or condition thatwould benefit from a decreased level of PD-L1 or a decreased bioactivityof PD-L1.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth divide and grow results in the formation ofmalignant tumors that invade neighboring tissues and may alsometastasize to distant parts of the body through the lymphatic system orbloodstream.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including Abs, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom a vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues.

An “immunoregulator” refers to a substance, an agent, a signalingpathway or a component thereof that regulates an immune response.“Regulating,” “modifying” or “modulating” an immune response refers toany alteration in a cell of the immune system or in the activity of suchcell. Such regulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunoregulators have been identified,some of which may have enhanced function in the cancer microenvironment.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down or preventing the onset, progression,development, severity or recurrence of a symptom, complication,condition or biochemical indicia associated with a disease.

“Administration” or “administering,” as used herein in the context ofanti-PD-L1 Adnectins, refers to introducing a PD-L1 Adnectin or PD-L1Adnectin-based probe or a labeled probe (also referred to as the“imaging agent”) described herein into a subject. Any route ofadministration is suitable, such as intravenous, oral, topical,subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal,nasal, introduction into the cerebrospinal fluid, or instillation intobody compartments can be used.

The term “therapeutically effective amount” refers to at least theminimal dose, but less than a toxic dose, of an agent which is necessaryto impart a therapeutic benefit to a subject.

As used herein, an “effective amount” refers to at least an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result.

As used herein, a “sufficient amount” refers to an amount sufficient toachieve the desired result.

As used herein, “positron emission tomography” or “PET” refers to anon-invasive, nuclear medicine technique that produces athree-dimensional image of tracer location in the body. The methoddetects pairs of gamma rays emitted indirectly by a positron-emittingradionuclide (tracer), which is introduced into the body on abiologically active molecule. PET imaging tools have a wide variety ofuses and aid in drug development both preclinically and clinically.Exemplary applications include direct visualization of in vivosaturation of targets; monitoring uptake in normal tissues to anticipatetoxicity or patient to patient variation; quantifying diseased tissue;tumor metastasis; and monitoring drug efficacy over time, or resistanceover time.

Overview

Provided herein are polypeptides that bind to human PD-L1 and can becoupled to heterologous molecule(s), such as a radiolabel. Suchpolypeptides are useful, for example, for detecting PD-L1 in a sample ortissue (e.g., a tissue, such as a cancer tissue that selectivelyexpresses PD-L1) for diagnostic assays.

The invention is based on the development of a non-invasive clinicalimaging agent that allows for whole body visualization of a patient'sPD-L1 expression. In certain embodiments, single day “virtual biopsies”of a patient's whole body are performed to monitor and localize PD-L1expression levels. PD-L1 imaging agents described herein may be used toprovide a high contrast whole-body virtual biopsy in a single day.

I. Fibronectin-Based Scaffolds

Fn3 refers to a type III domain from fibronectin. An Fn3 domain issmall, monomeric, soluble, and stable. It lacks disulfide bonds and,therefore, is stable under reducing conditions. The overall structure ofFn3 resembles the immunoglobulin fold. Fn3 domains comprise, in orderfrom N-terminus to C-terminus, a beta or beta-like strand, A; a loop,AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-likestrand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a betaor beta-like strand, E; a loop, EF; a beta or beta-like strand, F; aloop, FG; and a beta or beta-like strand, G. The seven antiparallelβ-strands are arranged as two beta sheets that form a stable core, whilecreating two “faces” composed of the loops that connect the beta orbeta-like strands. Loops AB, CD, and EF are located at one face (“thesouth pole”) and loops BC, DE, and FG are located on the opposing face(“the north pole”). There are at least 15 different Fn3 modules in humanFibronectin, and while the sequence homology between the modules is low,they all share a high similarity in tertiary structure.

Described herein are anti-PD-L1 Adnectins comprising an Fn3 domain inwhich one or more of the solvent accessible loops has been randomized ormutated. In certain embodiments, the Fn3 domain is an Fn3 domain derivedfrom the wild-type tenth module of the human fibronectin type III domain(¹⁰Fn3):

(SEQ ID NO: 1) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(94 amino acids; AB, CD, and EF loops are underlined; the core ¹⁰Fn3domain begins with amino acid 9 (“E”) and ends with amino acid 94 (“T”)and corresponds to an 86 amino acid polypeptide). The core wild-typehuman ¹⁰Fn3 domain is set forth in SEQ ID NO: 2.

In certain embodiments, the non-ligand binding sequences of ¹⁰Fn3, i.e.,the “¹⁰Fn3 scaffold”, may be altered provided that the ¹⁰Fn3 retainsligand binding function and/or structural stability. A variety of mutant¹⁰Fn3 scaffolds have been reported. In one aspect, one or more of Asp 7,Glu 9, and Asp 23 is replaced by another amino acid, such as, forexample, a non-negatively charged amino acid residue (e.g., Asn, Lys,etc.). A variety of additional alterations in the ¹⁰Fn3 scaffold thatare either beneficial or neutral have been disclosed. See, for example,Batori et al., Protein Eng., 15(12):1015-1020 (December 2002); Koide etal., Biochemistry, 40(34):10326-10333 (Aug. 28, 2001).

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO: 1, the AB loopcorresponds to residues 14-17, the BC loop corresponds to residues23-31, the CD loop corresponds to residues 37-47, the DE loopcorresponds to residues 51-56, the EF loop corresponds to residues63-67, and the FG loop corresponds to residues 76-87.

Accordingly, in certain embodiments, the anti-PD-L1 Adnectin describedherein is an ¹⁰Fn3 polypeptide that is at least 40%, 50%, 60%, 65%, 70%,75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3 domain, shown in SEQID NO: 1, or its core sequence, as shown in SEQ ID NO: 2. Much of thevariability will generally occur in one or more of the loops or one ormore of the beta strands or N- or C-terminal regions. Each of the betaor beta-like strands of a ¹⁰Fn3 polypeptide may consist essentially ofan amino acid sequence that is at least 80%, 85%, 90%, 95% or 100%identical to the sequence of a corresponding beta or beta-like strand ofSEQ ID NO: 1 or 2, provided that such variation does not disrupt thestability of the polypeptide in physiological conditions.

In certain embodiments, the invention provides an anti-human PD-L1Adnectin comprising a tenth fibronectin type III (¹⁰Fn3) domain, whereinthe ¹⁰Fn3 domain comprises a loop, AB; a loop, BC; a loop, CD; a loop,DE; a loop EF; and a loop FG; and has at least one loop selected fromloop BC, DE, and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human ¹⁰Fn3 domain. In someembodiments, the anti-PD-L1 Adnectins described herein comprise a ¹⁰Fn3domain comprising an amino acid sequence at least 80%, 85%, 90%, 95%,98%, 99% or 100% identical to the non-loop regions of SEQ ID NO: 1 or 2,wherein at least one loop selected from BC, DE, and FG is altered. Incertain embodiments, the BC and FG loops are altered, in certainembodiments, the BC and DE loops are altered, in certain embodiments,the DE and FG loops are altered, and in certain embodiments, the BC, DE,and FG loops are altered, i.e., the ¹⁰Fn3 domains comprise non-naturallyoccurring loops. In certain embodiments, the AB, CD and/or the EF loopsare altered. By “altered” is meant one or more amino acid sequencealterations relative to a template sequence (corresponding humanfibronectin domain) and includes amino acid additions, deletions,substitutions or a combination thereof. Altering an amino acid sequencemay be accomplished through intentional, blind, or spontaneous sequencevariation, generally of a nucleic acid coding sequence, and may occur byany technique, for example, PCR, error-prone PCR, or chemical DNAsynthesis.

In certain embodiments, one or more loops selected from BC, DE, and FGmay be extended or shortened in length relative to the correspondinghuman fibronectin loop. In some embodiments, the length of the loop maybe extended by 2-25 amino acids. In some embodiments, the length of theloop may be decreased by 1-11 amino acids. To optimize antigen binding,therefore, the length of a loop of ¹⁰Fn3 may be altered in length aswell as in sequence to obtain the greatest possible flexibility andaffinity in antigen binding.

In certain embodiments, the polypeptide comprises a Fn3 domain thatcomprises an amino acid sequence of the non-loop regions that is atleast 80, 85, 90, 95, 98, 99, or 100% identical to the non-loop regionsof SEQ ID NO: 1 or 2, wherein at least one loop selected from BC, DE,and FG is altered. In some embodiments, the altered BC loop has up to 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, up to 1, 2, 3,or 4 amino acid deletions, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid insertions, or a combination thereof.

In some embodiments, one or more residues of the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ IDNO: 1) may be substituted so as to disrupt integrin binding. In someembodiments, the FG loop of the polypeptides provided herein does notcontain an RGD integrin binding site. In one embodiment, the RGDsequence is replaced by a polar amino acid-neutral amino acid-acidicamino acid sequence (in the N-terminal to C-terminal direction). In someembodiments, the RGD sequence is replaced with SGE. In one embodiment,the RGD sequence is replaced with RGE.

In certain embodiments, the fibronectin based scaffold protein comprisesa ¹⁰Fn3 domain that is defined generally by following the sequence:

(SEQ ID NO: 3) EVVAA(Z)_(a) LLISW(Z)_(x) YRITY(Z)_(b) FTV(Z)_(y)ATISGL(Z)_(c) YTITVYA (Z)_(z) ISINYRTwherein the AB loop is represented by (Z)_(a), the CD loop isrepresented by (Z)_(b), the EF loop is represented by (Z)_(c), the BCloop is represented by (Z)_(x), the DE loop is represented by (Z)_(y),and the FG loop is represented by (Z)_(z). Z represents any amino acidand the subscript following the Z represents an integer of the number ofamino acids. In particular, a may be anywhere from 1-15, 2-15, 1-10,2-10, 1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino acids; and b,c, x, y and z may each independently be anywhere from 2-20, 2-15, 2-10,2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7amino acids. The sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 substitutions, deletions or additions acrossall 7 scaffold regions relative to the corresponding amino acids shownin SEQ ID NO: 1 or 2. In certain embodiments, the sequences of the betastrands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservativesubstitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 1 or 2. In certainembodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues.

In certain embodiments, the anti-PD-L1 Adnectins described herein arebased on a ¹⁰Fn3 scaffold and are defined generally by the sequence:

(SEQ ID NO: 4) EVVAATPTSLLISW(Z)_(x)YRITYGETGGNSPVQEFTV(Z)_(y)ATISGLKPGVDYTITVYA(Z)_(z)ISINYRT.wherein the BC loop is represented by (Z)_(x), the DE loop isrepresented by (Z)_(y), and the FG loop is represented by (Z)_(z). Zrepresents any amino acid and the subscript following the Z representsan integer of the number of amino acids. In particular, x, y and z mayeach independently be anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15,5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. Inpreferred embodiments, x is 11 amino acids, y is 6 amino acids, and z is12 amino acids. The sequences of the beta strands may have anywhere from0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to3, from 0 to 2, or from 0 to 1 substitutions, deletions or additionsacross all 7 scaffold regions relative to the corresponding amino acidsshown in SEQ ID NO: 1 or 2. In certain embodiments, the sequences of thebeta strands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6,from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1conservative substitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 1 or 2. In certainembodiments, the core amino acid residues, e.g., outside one or moreloops, are fixed and any substitutions, conservative substitutions,deletions or additions occur at residues other than the core amino acidresidues.

In certain embodiments, an anti-PD-L1 Adnectin may comprise the sequenceas set forth in SEQ ID NO: 3 or 4, wherein at least one of BC, DE, andFG loops as represented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively,are altered. As described above, amino acid residues corresponding toresidues 23-31, 51-56, and 76-87 of SEQ ID NO: 1 define the BC, DE, andFG loops, respectively. However, it should be understood that not everyresidue within the loop region needs to be modified in order to achievea ¹⁰Fn3 binder having strong affinity for a desired target (e.g.,PD-L1).

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs:21, 22, and 23, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 21, 22,and 23, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs:36, 37, and 38, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 36, 37,and 38, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs:51, 52, and 53, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 51, 52,and 53, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs:66, 67, and 68, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 66, 67,and 68, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs: 6,7, and 8, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 6, 7,and 8, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs:81, 82, and 83, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 81, 82,and 83, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, have aminoacid sequences at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the BC, DE or FG loop sequences set forth in SEQ ID NOs:97, 98, and 99, respectively. In certain embodiments, an anti-PD-L1Adnectin comprises the sequence set forth in SEQ ID NO: 3 or 4, whereinBC, DE and FG loops as represented by (Z)_(x), (Z)_(y), and (Z)_(z),respectively, comprise BC, DE, and FG loops having the amino acidsequences of SEQ ID NOs: 97, 98, and 99, respectively.

In certain embodiments, an anti-PD-L1 Adnectin comprises the sequenceset forth in SEQ ID NO: 3 or 4, wherein BC, DE and FG loops asrepresented by (Z)_(x), (Z)_(y), and (Z)_(z), respectively, comprise BC,DE, and FG loops having the amino acid sequences of SEQ ID NOs: 113,114, and 115, respectively; SEQ ID NOs: 124, 125 and 126, respectively;SEQ ID NOs: 135, 136 and 137, respectively; SEQ ID NOs: 146, 147 and148, respectively; SEQ ID NOs: 157, 158 and 159, respectively; SEQ IDNOs: 168, 169 and 170, respectively; SEQ ID NOs: 179, 180 and 181,respectively; SEQ ID NOs: 190, 191 and 192, respectively; SEQ ID NOs:201, 202 and 203, respectively; SEQ ID NOs: 212, 213 and 214,respectively; SEQ ID NOs: 223, 224 and 225, respectively; SEQ ID NOs:234, 235, and 236, respectively; SEQ ID NOs: 245, 246 and 247,respectively; SEQ ID NOs: 256, 257 and 258, respectively; SEQ ID NOs:267, 268 and 269, respectively; SEQ ID NOs: 278, 279 and 280,respectively; SEQ ID NOs: 289, 290 and 291, respectively; SEQ ID NOs:300, 301 and 302, respectively; SEQ ID NOs: 311, 312 and 313,respectively; SEQ ID NOs: 322, 323 and 324, respectively; SEQ ID NOs:333, 334 and 335, respectively; SEQ ID NOs: 344, 345 and 346,respectively; SEQ ID NOs: 355, 356 and 357, respectively; SEQ ID NOs:366, 367 and 368, respectively; SEQ ID NOs: 377, 378 and 379,respectively; SEQ ID NOs: 388, 389 and 390 respectively; SEQ ID NOs:399, 400 and 401, respectively; SEQ ID NOs: 410, 411 and 412,respectively; SEQ ID NOs: 421, 422 and 423, respectively; SEQ ID NOs:432, 433 and 434 respectively; SEQ ID NOs: 443, 444 and 445,respectively; SEQ ID NOs: 454, 455 and 456, respectively; SEQ ID NOs:465, 466 and 467, respectively; SEQ ID NOs: 476, 477 and 478,respectively; SEQ ID NOs: 487, 488 and 489, respectively; SEQ ID NOs:498, 499 and 500, respectively; SEQ ID NOs: 509, 510 and 511,respectively; SEQ ID NOs: 520, 521 and 522, respectively; SEQ ID NOs:531, 530 and 531, respectively; SEQ ID NOs: 542, 543 and 544,respectively; SEQ ID NOs: 553, 554 and 555, respectively; or SEQ ID NOs:564, 565 and 566, respectively. The scaffold regions of such anti-PD-L1Adnectins may comprise anywhere from 0 to 20, from 0 to 15, from 0 to10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 substitutions, conservative substitutions,deletions or additions relative to the scaffold amino acids residues ofSEQ ID NO: 4. Such scaffold modifications may be made, so long as theanti-PD-L1 Adnectin is capable of binding PD-L1 with a desired K_(D).

In certain embodiments, the BC loop of the anti-PD-L1 Adnectin comprisesan amino acid sequence selected from the group consisting of: 6, 21, 36,51, 66, 81, and 97.

In certain embodiments, the DE loop of the anti-PD-L1 Adnectin comprisesan amino acid sequence selected from the group consisting of: 7, 22, 37,52, 67, 82, and 98.

In certain embodiments, the FG loop of the anti-PD-L1 Adnectin comprisesan amino acid sequence selected from the group consisting of: 8, 23, 38,53, 68, 83, and 99.

In certain embodiments, the anti-PD-L1 Adnectin comprises a BC, DE andFG loop amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,99% or 100% identical to any one of SEQ ID NOs: 6, 21, 36, 51, 66, 81,and 97; 7, 22, 37, 52, 67, 82, and 98; and 8, 23, 38, 53, 68, 83, and99, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises an amino acidsequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 5, 20, 35, 50, 65, 80, 96, 112, 123,134, 145, 156, 167, 178, 189, 200, 211, 222, 233, 244, 255, 266, 277,288, 299, 310, 321, 332, 343, 354, 365, 376, 387, 398, 409, 420, 431,442, 453, 464, 475, 486, 497, 508, 519, 530, 541, 552 and 563.

In certain embodiments, the anti-PD-L1 Adnectins described hereincomprise an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 5,20, 35, 50, 65, 80, or 96.

In certain embodiments, the anti-PD-L1 Adnectin comprises an amino acidsequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to any one of SEQ ID NOs: 9-15, 24-30, 39-45, 54-60, 6975,84-91, 100-107, 116-122, 127-133, 138-144, 150-155, 160-166, 171-177,182-188, 193-199, 204-210, 215-221, 227-232, 237-243, 248-254, 259-265,271-276, 291-287, 292-298, 303-309, 314-320, 325-331, 337-342, 347-353,358-364, 369-375, 380-386, 391-397, 402-408, 413-419, 424-430, 435-441,446-452, 457-463, 468-474, 479-485, 490-496, 501-507, 512-518, 523-529,534-540, 545-551, and 556-562. In certain embodiments, the anti-PD-L1Adnectins described herein comprise an amino acid sequence at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loopregions of any one of SEQ ID NOs: 9-15, 24-30, 39-45, 54-60, 6975,84-91, and 100-107.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 6, 7, and 8, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 21, 22, and 23, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 36, 37, and 38, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 51, 52, and 53, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 66, 67, and 68, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 81, 82, and 83, respectively.

In certain embodiments, the anti-PD-L1 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 97, 98, and 99, respectively.

In certain embodiments, BC, DE and/or FG loop amino acid sequencesdescribed herein (e.g., SEQ ID NOs: 6, 21, 36, 51, 66, 81, and 97; 7,22, 37, 52, 67, 82, and 98; and 8, 23, 38, 53, 68, 83, and 99,respectively) are grafted into non-¹⁰Fn3 domain protein scaffolds. Forinstance, one or more loop amino acid sequences is exchanged for orinserted into one or more CDR loops of an antibody heavy or light chainor fragment thereof. In some embodiments, the protein domain into whichone or more amino acid loop sequences are exchanged or insertedincludes, but is not limited to, consensus Fn3 domains (Centocor, US),ankyrin repeat proteins (Molecular Partners AG, Zurich Switzerland),domain antibodies (Domantis, Ltd, Cambridge, Mass.), single domaincamelid nanobodies (Ablynx, Belgium), lipocalins (e.g., anticalins;Pieris Proteolab AG, Freising, Germany), Avimers (Amgen, Calif.),affibodies (Affibody AG, Sweden), ubiquitin (e.g., affilins; ScilProteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd,Allschwil, Switzerland), helical bundle scaffolds (e.g. alphabodies,Complix, Belgium), Fyn SH3 domains (Covagen AG, Switzerland), oratrimers (Anaphor, Inc., CA).

In certain embodiments, the amino acid sequences of the N-terminaland/or C-terminal regions of the polypeptides provided herein may bemodified by deletion, substitution or insertion relative to the aminoacid sequences of the corresponding regions of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1 or 2). The ¹⁰Fn3 domains generally begin with aminoacid number 1 of SEQ ID NO: 1. However, domains with amino aciddeletions are also encompassed by the invention. Additional sequencesmay also be added to the N- or C-terminus of a ¹⁰Fn3 domain having theamino acid sequence of SEQ ID NO: 1 or 2. For example, in someembodiments, the N-terminal extension consists of an amino acid sequenceselected from the group consisting of: M, MG, and G. In certainembodiments, an MG sequence may be placed at the N-terminus of the ¹⁰Fn3defined by SEQ ID NO: 1. The M will usually be cleaved off, leaving a Gat the N-terminus. In addition, an M, G or MG may also be placedN-terminal to any of the N-terminal extensions shown in Table 3.

In exemplary embodiments, an alternative N-terminal region having from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in lengthcan be added to the N-terminal region of SEQ ID NO: 1 or 2 or anyadnectin set forth in Table 3. Exemplary alternative N-terminal regionsinclude (represented by the single letter amino acid code) M, MG, G,MGVSDVPRDL (SEQ ID NO: 574) and GVSDVPRDL (SEQ ID NO: 575). Othersuitable alternative N-terminal regions, which may be linked, e.g., tothe N-terminus of an adnectin core sequence, include, for example,X_(n)SDVPRDL (SEQ ID NO: 576), X_(n)DVPRDL (SEQ ID NO: 577), X_(n)VPRDL(SEQ ID NO: 578), X_(n)PRDL (SEQ ID NO: 579) X_(n)RDL (SEQ ID NO: 580),X_(n)DL (SEQ ID NO: 581), or X_(n)L, wherein n=0, 1 or 2 amino acids,wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly. When aMet-Gly sequence is added to the N-terminus of a ¹⁰Fn3 domain, the Mwill usually be cleaved off, leaving a G at the N-terminus. In someembodiments, the alternative N-terminal region comprises the amino acidsequence MASTSG (SEQ ID NO: 582).

In exemplary embodiments, an alternative C-terminal region having from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in lengthcan be added to the C-terminal region of SEQ ID NO: 1 or 2 or anyadnectin set forth in Table 3. Specific examples of alternativeC-terminal region sequences include, for example, polypeptidescomprising, consisting essentially of, or consisting of, EIEK (SEQ IDNO: 584), EGSGC (SEQ ID NO: 585), EIEKPCQ (SEQ ID NO: 586), EIEKPSQ (SEQID NO: 587), EIEKP (SEQ ID NO: 588), EIEKPS (SEQ ID NO: 589), or EIEKPC(SEQ ID NO: 590). In some embodiments, the alternative C-terminal regioncomprises EIDK (SEQ ID NO: 591), and in particular embodiments, thealternative C-terminal region is either EIDKPCQ (SEQ ID NO: 592) orEIDKPSQ (SEQ ID NO: 593). Additional suitable alternative C-terminalregions are set forth in SEQ ID NOs: 594-618.

In certain embodiments, an Adnectin is linked to a C-terminal extensionsequence that comprises E and D residues, and may be between 8 and 50,10 and 30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. Insome embodiments, tail sequences include ED-based linkers in which thesequence comprises tandem repeats of ED. In exemplary embodiments, thetail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 EDrepeats. In certain embodiments, the ED-based tail sequences may alsoinclude additional amino acid residues, such as, for example: EI, EID,ES, EC, EGS, and EGC. Such sequences are based, in part, on knownAdnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 593), in whichresidues D and K have been removed. In exemplary embodiments, theED-based tail comprises an E, I or E1 residues before the ED repeats.

In certain embodiments, the N- or C-terminal extension sequences arelinked to the anti-PD-L1 Adnectin sequences with known linker sequences(e.g., SEQ ID NOs: 629-678 in Table 3). In some embodiments, sequencesmay be placed at the C-terminus of the ¹⁰Fn3 domain to facilitateattachment of a pharmacokinetic moiety. For example, a cysteinecontaining linker such as GSGC (SEQ ID NO: 638) may be added to theC-terminus to facilitate site directed PEGylation on the cysteineresidue.

In certain embodiments, an alternative C-terminal moiety, which can belinked to the C-terminal amino acids RT (i.e., amino acid 94) comprisesthe amino acids P_(m)X_(n), wherein P is proline, X is any amino acid, mis an integer that is at least 1 and n is 0 or an integer that is atleast 1. In certain embodiments, the alternative C-terminal moietycomprises the amino acids PC. In certain embodiments, the alternativeC-terminal moiety comprises the amino acids PI, PC, PID, PIE, PIDK (SEQID NO: 605), PIEK (SEQ ID NO: 606), PIDKP (SEQ ID NO: 607), PIEKP (SEQID NO: 608), PIDKPS (SEQ ID NO: 609), PIEKPS (SEQ ID NO: 610), PIDKPC(SEQ ID NO: 611), PIEKPC (SEQ ID NO: 612), PIDKPSQ (SEQ ID NO: 613),PIEKPSQ (SEQ ID NO: 614), PIDKPCQ (SEQ ID NO: 615), PIEKPCQ (SEQ ID NO:616), PHHHHHH (SEQ ID NO: 617), and PCHHHHHH (SEQ ID NO: 618). Exemplaryanti-PD-L1 Adnectins having PC at their C-terminus are provided in theExamples and Table 3.

In certain embodiments, the Adnectins described herein have a 6×his tail(SEQ ID NO: 619).

In certain embodiments, the fibronectin based scaffold proteins comprisea ¹⁰Fn3 domain having both an alternative N-terminal region sequence andan alternative C-terminal region sequence, and optionally a 6×his tail.

II. Biological Properties of Anti-PD-L1 Adnectins

Provided herein are adnectins that bind to human PD-L1 with a KD of 10nM, 1 nM, 0.5 nM, 0.1 nM or less, as determined, e.g., by SPR (Biacore)and exhibit one or more of the following properties:

-   1. Inhibition of the interaction between human PD-L1 and human PD-1    by at least 50%, 70%, 80%, 90% or more, as determined, e.g., by flow    cytometry, e.g., using a human PD-1Fc protein and human PD-L1    positive cells, such as L2987 cells;-   2. Inhibition of the binding of human CD80 (B7-1) to human PD-L1 by    at least 50%, 70%, 80%, 90% or more, as determined, e.g., in an    ELISA assay or by SPR (Biacore);-   3. Inhibition of the binding of the anti-PD-L1 antibody 12A4    (described, e.g., in U.S. Pat. No. 7,943,743) to human PD-L1 by at    least 50%, 70%, 80%, 90% or more, as determined, e.g., in an ELISA    assay or by SPR (Biacore); and-   4. Inhibit cell proliferation in a mixed lymphocyte reaction (MLR).

In certain embodiments, an anti-PD-L1 adnectin binds to human PD-L1 witha KD of 1 nM or less and exhibits each one of properties 1-4. In certainembodiments, an anti-PD-L1 adnectin binds to human PD-L1 with a KD of0.1 nM or less and exhibits each one of properties 1-4.

Provided herein are adnectins that comprise an amino acid sequence thatis at least 70%, 80%, 90%, 95%, 97%, 98% or 99% identical to ananti-PD-L1 adnectin described herein or a portion thereof (e.g., the BC,DE and FG loops), bind to human PD-L1 with a KD of 10 nM, 1 nM, 0.5 nM,0.1 nM or less, as determined, e.g., by SPR (Biacore) and exhibit one ormore of the following properties:

-   1. Inhibition of the interaction between human PD-L1 and human PD-1    by at least 50%, 70%, 80%, 90% or more, as determined, e.g., by flow    cytometry, e.g., using a human PD-1Fc protein and human PD-L1    positive cells, such as L2987 cells;-   2. Inhibition of the binding of human CD80 (B7-1) to human PD-L1 by    at least 50%, 70%, 80%, 90% or more, as determined, e.g., in an    ELISA assay or by SPR (Biacore);-   3. Inhibition of the binding of the anti-PD-L1 antibody 12A4 to    human PD-L1 by at least 50%, 70%, 80%, 90% or more, as determined,    e.g., in an ELISA assay or by SPR (Biacore); and-   4. Inhibit cell proliferation in a mixed lymphocyte reaction (MLR).

In certain embodiments, an anti-PD-L1 adnectin comprises an amino acidsequence that is at least 70%, 80%, 90%, 95%, 97%, 98% or 99% identicalto an anti-PD-L1 adnectin described herein or a portion thereof (e.g.,the BC, DE and FG loops), binds to human PD-L1 with a KD of 1 nM or lessand exhibits each one of properties 1-4. In certain embodiments, ananti-PD-L1 adnectin comprises an amino acid sequence that is at least70%, 80%, 90%, 95%, 97%, 98% or 99% identical to an anti-PD-L1 adnectindescribed herein or a portion thereof (e.g., the BC, DE and FG loops),binds to human PD-L1 with a KD of 0.1 nM or less and exhibits each oneof properties 1-4.

In certain embodiments, the anti-PD-L1 Adnectins compete (e.g.,cross-compete) for binding to PD-L1 with the particular anti-PD-L1Adnectins described herein. Such competing Adnectins can be identifiedbased on their ability to competitively inhibit binding to PD-L1 ofAdnectins described herein in standard PD-L1 binding assays. Forexample, standard ELISA assays can be used in which a recombinant PD-L1protein is immobilized on the plate, one of the Adnectins isfluorescently labeled and the ability of non-labeled Adnectins tocompete off the binding of the labeled Adnectin is evaluated.

In certain embodiments, a competitive ELISA format can be performed todetermine whether two anti-PD-L1 Adnectins bind overlapping Adnectinbinding sites on PD-L1. In one format, Adnectin #1 is coated on a plate,which is then blocked and washed. To this plate is added either PD-L1alone, or PD-L1 pre-incubated with a saturating concentration ofAdnectin #2. After a suitable incubation period, the plate is washed andprobed with a polyclonal anti-PD-L1 antibody, such as a biotinylatedanti-PD-L1 polyclonal antibody, followed by detection withstreptavidin-HRP conjugate and standard tetramethylbenzidine developmentprocedures. If the OD signal is the same with or without preincubationwith Adnectin #2, then the two Adnectins bind independently of oneanother, and their Adnectin binding sites do not overlap. If, however,the OD signal for wells that received PD-L1/Adnectin#2 mixtures is lowerthan for those that received PD-L1 alone, then binding of Adnectin #2 isconfirmed to block binding of Adnectin #1 to PD-L1.

Alternatively, a similar experiment is conducted by surface plasmonresonance (SPR, e.g., BIAcore). Adnectin #1 is immobilized on an SPRchip surface, followed by injections of either PD-L1 alone or PD-L1pre-incubated with a saturating concentration of Adnectin #2. If thebinding signal for PD-L1/Adnectin#2 mixtures is the same or higher thanthat of PD-L1 alone, then the two Adnectins bind independently of oneanother, and their Adnectin binding sites do not overlap. If, however,the binding signal for PD-L1/Adnectin#2 mixtures is lower than thebinding signal for PD-L1 alone, then binding of Adnectin #2 is confirmedto block binding of Adnectin #1 to PD-L1. A feature of these experimentsis the use of saturating concentrations of Adnectin #2. If PD-L1 is notsaturated with Adnectin #2, then the conclusions above do not hold.Similar experiments can be used to determine if any two PD-L1 bindingproteins bind to overlapping Adnectin binding sites.

Both assays exemplified above may also be performed in the reverse orderwhere Adnectin#2 is immobilized and PD-L1-Adnectin#1 are added to theplate. Alternatively, Adnectin #1 and/or #2 can be replaced with amonoclonal antibody and/or soluble receptor-Fc fusion protein.

In certain embodiments, competition can be determined using a HTRFsandwich assay.

In certain embodiments, the competing Adnectin is an Adnectin that bindsto the same Adnectin binding site on PD-L1 as a particular anti-PD-L1Adnectin described herein. Standard mapping techniques, such as proteasemapping, mutational analysis, HDX-MS, x-ray crystallography and2-dimensional nuclear magnetic resonance, can be used to determinewhether an Adnectin binds to the same Adnectin binding site or epitopeas a reference Adnectin (see, e.g., Epitope Mapping Protocols in Methodsin Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). An epitope isdefined by the method used to locate it. For example, in certainembodiments, a PD-L1 adnectin or antibody binds to the same epitope asthat of one of the PD-L1 adnectins described herein, as determined byHDX-MS or as determined by X-ray crystallography.

Candidate competing anti-PD-L1 Adnectins can inhibit the binding ofanti-PD-L1 Adnectins described herein to PD-L1 by at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, or at least 99% and/or their binding is inhibited by at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% by anti-PD-L1 Adnectins. The % competitioncan be determined using the methods described above.

Provided herein are adnectins that bind to human PD-L1 with a KD of 10nM, 1 nM, 0.5 nM, 0.1 nM or less, as determined, e.g., by SPR (Biacore)and exhibit one or more of the following properties:

-   1. Inhibition of the interaction between human PD-L1 and human PD-1    by at least 50%, 70%, 80%, 90% or more, as determined, e.g., by flow    cytometry, e.g., using a human PD-1Fc protein and human PD-L1    positive cells, such as L2987 cells;-   2. Inhibition of the binding of human CD80 (B7-1) to human PD-L1 by    at least 50%, 70%, 80%, 90% or more, as determined, e.g., in an    ELISA assay or by SPR (Biacore);-   3. Inhibition of the binding of the anti-PD-L1 antibody 12A4 to    human PD-L1 by at least 50%, 70%, 80%, 90% or more, as determined,    e.g., in an ELISA assay or by SPR (Biacore);-   4. Inhibit cell proliferation in a mixed lymphocyte reaction (MLR);    and-   5. Compete with an anti-PD-L1 antibody described herein for binding    to human PD-L1.

In certain embodiments, an anti-PD-L1 adnectin binds to human PD-L1 witha KD of 1 nM or less and exhibits each one of properties 1-5. In certainembodiments, an anti-PD-L1 adnectin binds to human PD-L1 with a KD of0.1 nM or less and exhibits each one of properties 1-5.

Provided herein are adnectins that comprise an amino acid sequence thatis at least 70%, 80%, 90%, 95%, 97%, 98% or 99% identical to ananti-PD-L1 adnectin described herein or a portion thereof (e.g., the BC,DE and FG loops), bind to human PD-L1 with a KD of 10 nM, 1 nM, 0.5 nM,0.1 nM or less, as determined, e.g., by SPR (Biacore) and exhibit one ormore of the following properties:

-   1. Inhibition of the interaction between human PD-L1 and human PD-1    by at least 50%, 70%, 80%, 90% or more, as determined, e.g., by flow    cytometry, e.g., using a human PD-1Fc protein and human PD-L1    positive cells, such as L2987 cells;-   2. Inhibition of the binding of human CD80 (B7-1) to human PD-L1 by    at least 50%, 70%, 80%, 90% or more, as determined, e.g., in an    ELISA assay or by SPR (Biacore);-   3. Inhibition of the binding of the anti-PD-L1 antibody 12A4 to    human PD-L1 by at least 50%, 70%, 80%, 90% or more, as determined,    e.g., in an ELISA assay or by SPR (Biacore);-   4. Inhibit cell proliferation in a mixed lymphocyte reaction (MLR);    and-   5. Compete with an anti-PD-L1 antibody described herein for binding    to human PD-L1.

In certain embodiments, an anti-PD-L1 adnectin comprises an amino acidsequence that is at least 70%, 80%, 90%, 95%, 97%, 98% or 99% identicalto an anti-PD-L1 adnectin described herein or a portion thereof (e.g.,the BC, DE and FG loops), binds to human PD-L1 with a KD of 1 nM or lessand exhibits each one of properties 1-5. In certain embodiments, ananti-PD-L1 adnectin comprises an amino acid sequence that is at least70%, 80%, 90%, 95%, 97%, 98% or 99% identical to an anti-PD-L1 adnectindescribed herein or a portion thereof (e.g., the BC, DE and FG loops),binds to human PD-L1 with a KD of 0.1 nM or less and exhibits each oneof properties 1-5.

III. Fusions, Including Pharmacokinetic Moieties

In certain embodiments, the anti-PD-L1 Adnectins desirably have a shorthalf-life, for example, when used in diagnostic imaging.

Alternatively, e.g., for therapeutic purposes, the anti-PD-L1 Adnectinsdescribed herein further comprise a pharmacokinetic (PK) moiety.Improved pharmacokinetics may be assessed according to the perceivedtherapeutic need. Often it is desirable to increase bioavailabilityand/or increase the time between doses, possibly by increasing the timethat a protein remains available in the serum after dosing. In someinstances, it is desirable to improve the continuity of the serumconcentration of the protein over time (e.g., decrease the difference inserum concentration of the protein shortly after administration andshortly before the next administration). The anti-PD-L1 Adnectin may beattached to a moiety that reduces the clearance rate of the polypeptidein a mammal (e.g., mouse, rat, or human) by greater than two-fold,greater than three-fold, greater than four-fold or greater thanfive-fold relative to the unmodified anti-PD-L1 Adnectin. Other measuresof improved pharmacokinetics may include serum half-life, which is oftendivided into an alpha phase and a beta phase. Either or both phases maybe improved significantly by addition of an appropriate moiety. Forexample, the PK moiety may increase the serum half-life of thepolypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domainalone.

Moieties that slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties (e.g.,polyethylene glycol), sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc and fragments and variants thereof,transferrin, or serum albumin). The anti-PD-L1 Adnectin may also befused to albumin or a fragment (portion) or variant of albumin asdescribed in U.S. Publication No. 2007/0048282, or may be fused to oneor more serum albumin binding Adnectin, as described herein.

Other PK moieties that can be used in the invention include thosedescribed in Kontermann et al., (Current Opinion in Biotechnology 2011;22:868-76), herein incorporated by reference. Such PK moieties include,but are not limited to, human serum albumin fusions, human serum albuminconjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb,ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based onthe three amino acids proline, alanine, and serine), carbohydrateconjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialicacid conjugates, and fatty acid conjugates.

Accordingly, in some embodiments the invention provides an anti-PD-L1Adnectin fused to a PK moiety that is a polymeric sugar. In someembodiments, the PK moiety is a polyethylene glycol moiety or an Fcregion. In some embodiments the PK moiety is a serum albumin bindingprotein such as those described in U.S. Publication Nos. 2007/0178082and 2007/0269422. In some embodiments the PK moiety is human serumalbumin. In some embodiments, the PK moiety is transferrin.

In some embodiments, the PK moiety is linked to the anti-PD-L1 Adnectinvia a polypeptide linker. Exemplary polypeptide linkers includepolypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2amino acids. Suitable linkers for joining the Fn3 domains are thosewhich allow the separate domains to fold independently of each otherforming a three dimensional structure that permits high affinity bindingto a target molecule. Specific examples of suitable linkers includeglycine-serine based linkers, glycine-proline based linkers,proline-alanine based linkers as well as any other linkers describedherein. In some embodiments, the linker is a glycine-proline basedlinker. These linkers comprise glycine and proline residues and may bebetween 3 and 30, 10 and 30, and 3 and 20 amino acids in length.Examples of such linkers include GPG, GPGPGPG (SEQ ID NO: 672) andGPGPGPGPGPG (SEQ ID NO: 673). In some embodiments, the linker is aproline-alanine based linker. These linkers comprise proline and alanineresidues and may be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18amino acids in length. Examples of such linkers include PAPAPA (SEQ IDNO: 674), PAPAPAPAPAPA (SEQ ID NO: 675) and PAPAPAPAPAPAPAPAPA (SEQ IDNO: 676). In some embodiments, the linker is a glycine-serine basedlinker. These linkers comprise glycine and serine residues and may bebetween 8 and 50, 10 and 30, and 10 and 20 amino acids in length.Examples of such linkers include GSGSGSGSGS ((GS)₅; SEQ ID NO: 662),GSGSGSGSGSGS ((GS)₆; SEQ ID NO: 663), GSGSGSGSGSGSGSGSGSGS ((GS)₁₀; SEQID NO: 677), GGGGSGGGGSGGGGSGGGGS ((G₄S)₄; SEQ ID NO: 678),GGGGSGGGGSGGGGSGGGGSGGGGS ((G₄S)₅; SEQ ID NO: 670), and GGGGSGGGGSGGGSG(SEQ ID NO: 671). In exemplary embodiments, the linker does not containany Asp-Lys (DK) pairs. A list of suitable linkers is provided in Table3.

Optimal linker length and amino acid composition may be determined byroutine experimentation in view of the teachings provided herein. Insome embodiments, an anti-PD-L1 Adnectin is linked, for example, to ananti-HSA Adnectin via a polypeptide linker having a protease site thatis cleavable by a protease in the blood or target tissue. Suchembodiments can be used to release an anti-PD-L1 Adnectin for betterdelivery or therapeutic properties or more efficient production.

Additional linkers or spacers, may be introduced at the N-terminus orC-terminus of a Fn3 domain between the Fn3 domain and the polypeptidelinker.

Polyethylene Glycol

In some embodiments, the anti-PD-L1 Adnectin comprises polyethyleneglycol (PEG). PEG is a well-known, water soluble polymer that iscommercially available or can be prepared by ring-opening polymerizationof ethylene glycol according to methods well known in the art (Sandlerand Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages138-161). The term “PEG” is used broadly to encompass any polyethyleneglycol molecule, without regard to size or to modification at an end ofthe PEG, and can be represented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH, where n is 20 to 2300 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl. PEG can contain furtherchemical groups which are necessary for binding reactions, which resultfrom the chemical synthesis of the molecule; or which act as a spacerfor optimal distance of parts of the molecule. In addition, such a PEGcan consist of one or more PEG side-chains which are linked together.PEGs with more than one PEG chain are called multiarmed or branchedPEGs. Branched PEGs are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462.

One or more PEG molecules may be attached at different positions on theprotein, and such attachment may be achieved by reaction with amines,thiols or other suitable reactive groups. The amine moiety may be, forexample, a primary amine found at the N-terminus of a polypeptide or anamine group present in an amino acid, such as lysine or arginine. Insome embodiments, the PEG moiety is attached at a position on thepolypeptide selected from the group consisting of: a) the N-terminus; b)between the N-terminus and the most N-terminal beta strand or beta-likestrand; c) a loop positioned on a face of the polypeptide opposite thetarget-binding site; d) between the C-terminus and the most C-terminalbeta strand or beta-like strand; and e) at the C-terminus.

PEGylation may be achieved by site-directed PEGylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere PEGylation preferentially occurs. In some embodiments, the proteinis modified to introduce a cysteine residue at a desired position,permitting site-directed PEGylation on the cysteine. Mutations may beintroduced into a protein coding sequence to generate cysteine residues.This might be achieved, for example, by mutating one or more amino acidresidues to cysteine. Preferred amino acids for mutating to a cysteineresidue include serine, threonine, alanine and other hydrophilicresidues. Preferably, the residue to be mutated to cysteine is asurface-exposed residue. Algorithms are well-known in the art forpredicting surface accessibility of residues based on primary sequenceor a protein. Alternatively, surface residues may be predicted bycomparing the amino acid sequences of binding polypeptides, given thatthe crystal structure of the framework, based on which bindingpolypeptides are designed and evolved, has been solved (see Himanen etal., Nature 2001; 414:933-8) and thus the surface-exposed residuesidentified. PEGylation of cysteine residues may be carried out using,for example, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, orPEG-orthopyridyl disulfide. In certain embodiments, a PEG is linked to aC-terminal cysteine, e.g., a cysteine that has been added to ananti-PD-L1 Adnectin, such as in the form of a “PC” extension, as furtherdescribed herein.

The PEG is typically activated with a suitable activating groupappropriate for coupling to a desired site on the polypeptide.PEGylation methods are well-known in the art and further described inZalipsky, S., et al., “Use of Functionalized Poly(Ethylene Glycols) forModification of Polypeptides” in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press,New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 16:157-182.

PEG may vary widely in molecular weight and may be branched or linear.Typically, the weight-average molecular weight of PEG is from about 100Daltons to about 150,000 Daltons. Exemplary weight-average molecularweights for PEG include about 5,000 Daltons, about 10,000 Daltons, about20,000 Daltons, about 40,000 Daltons, about 60,000 Daltons and about80,000 Daltons. In certain embodiments, the molecular weight of PEG is40,000 Daltons. Branched versions of PEG having a total molecular weightof any of the foregoing can also be used. In some embodiments, the PEGhas two branches. In some embodiments, the PEG has four branches. Insome embodiments, the PEG is a bis-PEG (NOF Corporation, DE-200MA), inwhich two Adnectins are conjugated (see, e.g., Example 1 and ATI-1341 ofTable 5).

Conventional separation and purification techniques known in the art canbe used to purify PEGylated anti-PD-L1 Adnectins, such as size exclusion(e.g., gel filtration) and ion exchange chromatography. Products mayalso be separated using SDS-PAGE. Products that may be separated includemono-, di-, tri-, poly- and un-PEGylated Adnectins, as well as free PEG.The percentage of mono-PEG conjugates can be controlled by poolingbroader fractions around the elution peak to increase the percentage ofmono-PEG in the composition. About 90% mono-PEG conjugates represent agood balance of yield and activity.

In some embodiments, the PEGylated anti-PD-L1 Adnectins will preferablyretain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% ofthe biological activity associated with the unmodified anti-PD-L1Adnectin. In some embodiments, biological activity refers to its abilityto bind to PD-L1, as assessed by K_(D), k_(on), or k_(off). In someembodiments, the PEGylated anti-PD-L1 Adnectin shows an increase inbinding to PD-L1 relative to unPEGylated anti-PD-L1 Adnectin.

Immunoglobulin Fc Domain (and Fragments)

In certain embodiments, the anti-PD-L1 Adnectin is fused to animmunoglobulin Fc domain, or a fragment or variant thereof. As usedherein, a “functional Fc region” is an Fc domain or fragment thereofwhich retains the ability to bind FcRn. In some embodiments, afunctional Fc region binds to FcRn, bud does not possess effectorfunction. The ability of the Fc region or fragment thereof to bind toFcRn can be determined by standard binding assays known in the art. Insome embodiments, the Fc region or fragment thereof binds to FcRn andpossesses at least one “effector function” of a native Fc region.Exemplary “effector functions” include C1q binding; complement dependentcytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an anti-PD-L1 Adnectin) and can be assessed using variousassays known in the art for evaluating such antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification. Preferably, the variant Fc region has at least one aminoacid substitution compared to a native sequence Fc region or to the Fcregion of a parent polypeptide, e.g., from about one to about ten aminoacid substitutions, and preferably from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. The variant Fc region herein will preferablypossess at least about 80% sequence identity with a native sequence Fcregion and/or with an Fc region of a parent polypeptide, and mostpreferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

In an exemplary embodiment, the Fc domain is derived from an IgG1subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) mayalso be used. Shown below is the sequence of a human IgG1 immunoglobulinFc domain:

(SEQ ID NO: 620) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The core hinge sequence is underlined, and the CH2 and CH3 regions arein regular text. It should be understood that the C-terminal lysine isoptional. Allotypes and mutants of this sequence may also be used. As isknown in the art, mutants can be designed to modulate a variety ofproperties of the Fc, e.g., ADCC, CDC or half-life.

In certain embodiments, the Fc region used in the anti-PD-L1 Adnectinfusion comprises a CH1 region. In certain embodiments, the Fc regionused in the anti-PD-L1 Adnectin fusion comprises CH2 and CH3 regions. Incertain embodiments, the Fc region used in the anti-PD-L1 Adnectinfusion comprises a CH2, CH3, and hinge region (e.g., as shown in SEQ IDNO: 620).

In certain embodiments, the “hinge” region comprises the core hingeresidues spanning positions 1-16 of SEQ ID NO: 620 (DKTHTCPPCPAPELLG;SEQ ID NO: 621) of the IgG1 Fc region. In certain embodiments, theanti-PD-L1 Adnectin-Fc fusion adopts a multimeric structure (e.g.,dimer) owing, in part, to the cysteine residues at positions 6 and 9 ofSEQ ID NO: 620 within the hinge region. Other suitable exemplary hingeregions are set forth in SEQ ID NOs: 622-626.

Adnectins

In certain embodiments the PK moiety is another Adnectin specific, forexample, to a serum protein (e.g., human serum albumin), as described inUS 2012/0094909, herein incorporated by reference in its entirety. OtherPK moieties that may be used with the Adnectins described herein aredescribed in Kontermann et al. (Current Opinion in Biotechnology 2011;22:868-76), as discussed supra.

In some embodiments, an anti-PD-L1 Adnectin may be directly orindirectly linked for example, to an anti-HSA Adnectin via a polymericlinker. Polymeric linkers can be used to optimally vary the distancebetween each component of the fusion to create a protein fusion with oneor more of the following characteristics: 1) reduced or increased sterichindrance of binding of one or more protein domains when binding to aprotein of interest, 2) increased protein stability or solubility, 3)decreased protein aggregation, and 4) increased overall avidity oraffinity of the protein.

In some embodiments, an anti-PD-L1 Adnectin is linked, for example, toan anti-HSA Adnectin, via a biocompatible polymer such as a polymericsugar. The polymeric sugar can include an enzymatic cleavage site thatis cleavable by an enzyme in the blood or target tissue. Suchembodiments can be used to release an anti-PD-L1 Adnectin for betterdelivery or therapeutic properties or more efficient production.

IV. Nucleic Acid-Protein Fusion Technology

In one aspect, the invention provides an Adnectin comprising fibronectintype III domains that binds PD-L1. One way to rapidly make and test Fn3domains with specific binding properties is the nucleic acid-proteinfusion technology of Adnexus, a Bristol-Myers Squibb R&D Company. Thisdisclosure utilizes the in vitro expression and tagging technology,termed ‘PROfusion’ which exploits nucleic acid-protein fusions (RNA- andDNA-protein fusions) to identify novel polypeptides and amino acidmotifs that are important for binding to proteins. Nucleic acid-proteinfusion technology is a technology that covalently couples a protein toits encoding genetic information. For a detailed description of theRNA-protein fusion technology and fibronectin-based scaffold proteinlibrary screening methods see Szostak et al., U.S. Pat. Nos. 6,258,558,6,261,804, 6,214,553, 6,281,344, 6,207,446, 6,518,018 and 6,818,418;Roberts et al., Proc. Natl. Acad. Sci., 1997; 94:12297-12302; and Kurzet al., Molecules, 2000; 5:1259-64, all of which are herein incorporatedby reference.

V. Vectors and Polynucleotides

Also included in the present disclosure are nucleic acid sequencesencoding any of the proteins described herein. As appreciated by thoseskilled in the art, because of third base degeneracy, almost every aminoacid can be represented by more than one triplet codon in a codingnucleotide sequence. In addition, minor base pair changes may result ina conservative substitution in the amino acid sequence encoded but arenot expected to substantially alter the biological activity of the geneproduct. Therefore, a nucleic acid sequence encoding a protein describedherein may be modified slightly in sequence and yet still encode itsrespective gene product. Certain exemplary nucleic acids encoding theanti-PD-L1 Adnectins and their fusions described herein include nucleicacids having the sequences set forth in SEQ ID NOs: 16-19, 31-34, 46-49,61-64, 76-79, 92-95, and 108-111.

Also contemplated are nucleic acid sequences that are at least 50%, suchas at least 55%, at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 16-19,31-34, 46-49, 61-64, 76-79, 92-95, and 108-111, and encode a proteinthat binds to PD-L1. In some embodiments, nucleotide substitutions areintroduced so as not to alter the resulting translated amino acidsequence.

Nucleic acids encoding any of the various proteins or polypeptidesdescribed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21,2003); Sinclair et al., Protein Expr Purif., 26(I):96-105 (October2002); Connell, N. D., Curr Opin. Biotechnol., 12(5):446-449 (October2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (September 1996);and Sharp et al., Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described in, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), orAusubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. Generally, the DNA encoding thepolypeptide is operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, viral, orinsect genes. Such regulatory elements include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding site, and sequencesthat control the termination of transcription and translation. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants is additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. An exemplary N-terminal leader sequence forproduction of polypeptides in a mammalian system is:METDTLLLWVLLLWVPGSTG (SEQ ID NO: 583), which is removed by the host cellfollowing expression.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., a yeast invertase leader, a factor leader (including Saccharomycesand Kluyveromyces alpha-factor leaders), or acid phosphatase leader, theC. albicans glucoamylase leader, or the signal sequence described inU.S. Pat. No. 5,631,144. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available. The DNA for such precursor regions maybe ligated in reading frame to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein described herein, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein described herein. Promoter sequences are known foreukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding protein described herein by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the peptide-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein described herein.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO 94/11026 and the expressionvector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude, but are not limited to, a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York(1985)), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides described herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

VI. Protein Production

Also described herein are cell lines that express an anti-PD-L1 Adnectinor fusion polypeptide thereof. Creation and isolation of cell linesproducing an anti-PD-L1 Adnectin can be accomplished using standardtechniques known in the art, such as those described herein.

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

Adnectins of the present invention can also be obtained in aglycosylatedform by producing the Adnectins in, e.g., prokaryotic cells (e.g., E.coli). Notably, aglycosylated forms of the Adnectins described hereinexhibit the same affinity, potency, and mechanism of action asglycosylated Adnectins when tested in vitro.

The host cells used to produce the proteins of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma)) aresuitable for culturing the host cells. In addition, many of the mediadescribed in Ham et al., Meth. Enzymol., 58:44 (1979), Barites et al.,Anal. Biochem., 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866,4,927,762, 4,560,655, 5,122,469, 6,048,728, 5,672,502, or U.S. Pat. No.RE 30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as Gentamycin drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Proteins described herein can also be produced using cell-freetranslation systems. For such purposes the nucleic acids encoding thepolypeptide must be modified to allow in vitro transcription to producemRNA and to allow cell-free translation of the mRNA in the particularcell-free system being utilized (eukaryotic such as a mammalian or yeastcell-free translation system or prokaryotic such as a bacterialcell-free translation system).

Proteins described herein can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2ndEdition, The Pierce Chemical Co., Rockford, Ill. (1984)). Modificationsto the protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, or preferablyat least 95% pure, and most preferably at least 98% pure. Regardless ofthe exact numerical value of the purity, the polypeptide is sufficientlypure for use as a pharmaceutical product.

High Throughput Protein Production (HTPP)

Selected binders cloned into the PET9d vector upstream of a HIS₆tag andare transformed into E. coli BL21 DE3 plysS cells and inoculated in 5 mlLB medium containing 50 μg/mL kanamycin in a 24-well format and grown at37° C. overnight. Fresh 5 ml LB medium (50 μg/mL kanamycin) cultures areprepared for inducible expression by aspiration of 200 μl from theovernight culture and dispensing it into the appropriate well. Thecultures are grown at 37° C. until A₆₀₀ 0.6-0.9. After induction with 1mM isopropyl-β-thiogalactoside (IPTG), the culture is expressed for 6hours at 30° C. and harvested by centrifugation for 10 minutes at 2750 gat 4° C.

Cell pellets (in 24-well format) are lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1mg/ml lysozyme, 30 μg/ml DNAse, 2 μg/ml aprotonin, pH 8.0) and shaken atroom temperature for 1-3 hours. Lysates are cleared and re-racked into a96-well format by transfer into a 96-well Whatman GF/D Unifilter fittedwith a 96-well, 1.2 ml catch plate and filtered by positive pressure.The cleared lysates are transferred to a 96-well Nickel orCobalt-Chelating Plate that had been equilibrated with equilibrationbuffer (50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole, pH 8.0) and areincubated for 5 min. Unbound material is removed by positive pressure.The resin is washed twice with 0.3 ml/well with Wash buffer #1 (50 mMNaH₂PO₄, 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0). Each wash isremoved by positive pressure. Prior to elution, each well is washed with50 μl Elution buffer (PBS+20 mM EDTA), incubated for 5 min, and thiswash is discarded by positive pressure. Protein is eluted by applying anadditional 100 μl of Elution buffer to each well. After a 30 minuteincubation at room temperature, the plate(s) are centrifuged for 5minutes at 200 g and eluted protein collected in 96-well catch platescontaining 5 μl of 0.5 M MgCl₂ added to the bottom of elution catchplate prior to elution. Eluted protein is quantified using a totalprotein assay with wild-type ¹⁰Fn3 domain as the protein standard.

Midscale Expression and Purification of Insoluble Fibronectin-BasedScaffold Protein Binders

For expression of insoluble clones, the clone(s), followed by theHIS₆tag, are cloned into a pET9d (EMD Bioscience, San Diego, Calif.)vector and are expressed in E. coli HMS174 cells. Twenty ml of aninoculum culture (generated from a single plated colony) is used toinoculate 1 liter of LB medium containing 50 μg/ml carbenicillin and 34μg/ml chloramphenicol. The culture is grown at 37° C. until A₆₀₀0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG)the culture is grown for 4 hours at 30° C. and is harvested bycentrifugation for 30 minutes at >10,000 g at 4° C. Cell pellets arefrozen at −80° C. The cell pellet is resuspended in 25 ml of lysisbuffer (20 mM aH2P0₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), ImM PMSF, pH 7.4) using an ULTRA-TURRAX®homogenizer (IKA works) on ice. Cell lysis is achieved by high pressurehomogenization (>18,000 psi) using a Model M-1 10S MICROFLUIDIZER®(Microfluidics). The insoluble fraction is separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The insoluble pellet recovered fromcentrifugation of the lysate is washed with 20 mM sodiumphosphate/500 mMNaCl, pH7.4. The pellet is resolubilized in 6.0M guanidine hydrochloridein 20 mM sodium phosphate/500M NaCl pH 7.4 with sonication followed byincubation at 37 degrees for 1-2 hours. The resolubilized pellet isfiltered to 0.45 μm and loaded onto a Histrap column equilibrated withthe 20 mM sodium phosphate/500 M NaCl/6.0 M guanidine pH 7.4 buffer.After loading, the column is washed for an additional 25 CV with thesame buffer. Bound protein is eluted with 50 mM Imidazole in 20 mMsodium phosphate/500 mM NaCl/6.0 M guan-HCl pH7.4. The purified proteinis refolded by dialysis against 50 mM sodium acetate/150 mM NaCl pH 4.5.

Midscale Expression and Purification of Soluble Fibronectin-BaseScaffold Protein Binders

For expression of soluble clones, the clone(s), followed by the HIS₆tag,are cloned into a pET9d (EMD Bioscience, San Diego, Calif.) vector andexpressed in E. coli HMS174 cells. Twenty ml of an inoculum culture(generated from a single plated colony) is used to inoculate 1 liter ofLB medium containing 50 μg/ml carbenicillin and 34 μg/mlchloramphenicol. The culture is grown at 37° C. until A₆₀₀ 0.6-1.0.After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), theculture is grown for 4 hours at 30° C. and harvested by centrifugationfor 30 minutes at >10,000 g at 4° C. Cell pellets are frozen at −80° C.The cell pellet is resuspended in 25 ml of lysis buffer (20 mM NaH₂PO₄,0.5 M NaCl, 1× Complete Protease Inhibitor Cocktail-EDTA free (Roche),ImM PMSF, pH 7.4) using an ULTRA-TURRAX® homogenizer (IKA works) on ice.Cell lysis is achieved by high pressure homogenization (>18,000 psi)using a Model M-1 10S MICROFLUIDIZER® (Microfluidics). The solublefraction is separated by centrifugation for 30 minutes at 23,300 g at 4°C. The supernatant is clarified via 0.45 μm filter. The clarified lysateis loaded onto a Histrap column (GE) pre-equilibrated with the 20 mMsodium phosphate/500M NaCl pH 7.4. The column is then washed with 25column volumes of the same buffer, followed by 20 column volumes of 20mM sodium phosphate/500 M NaCl/25 mM Imidazole, pH 7.4 and then 35column volumes of 20 mM sodium phosphate/500 M NaCl/40 mM Imidazole, pH7.4. Protein is eluted with 15 column volumes of 20 mM sodiumphosphate/500 M NaCl/500 mM Imidazole, pH 7.4, fractions are pooledbased on absorbance at A₂so and dialyzed against 1×PBS, 50 mM Tris, 150mM NaCl; pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH4.5. Any precipitate isremoved by filtering at 0.22 μm.

VII. Compositions

The present invention further provides compositions, such aspharmaceutical compositions and radiopharmaceutical compositions,comprising an anti-PD-L1 Adnectin or fusion proteins thereof describedherein, wherein the composition is essentially endotoxin free, or atleast contain no more than acceptable levels of endotoxins as determinedby the appropriate regulatory agency (e.g., FDA).

Compositions of the present invention can be in the form of a pill,tablet, capsule, liquid, or sustained release tablet for oraladministration; a liquid for intravenous, subcutaneous or parenteraladministration; or a gel, lotion, ointment, cream, or a polymer or othersustained release vehicle for local administration.

Methods well known in the art for making compositions are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Compositions for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate compositions (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the composition varies depending upon anumber of factors, including the dosage of the drug to be administered,the route of administration, and the purpose of the composition (e.g.,prophylactic, therapeutic, diagnostic).

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as Tween, PLURONIC™ or polyethylene glycol (PEG).

The polypeptides of the present invention may be optionally administeredas a pharmaceutically acceptable salt, such as non-toxic acid additionsalts or metal complexes that are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

The active ingredients may also be entrapped in a microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the proteins described herein, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. While encapsulated proteins described herein may remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Compositions of the present invention for oral use include tabletscontaining the active ingredient(s) in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients may be, forexample, inert diluents or fillers (e.g., sucrose and sorbitol),lubricating agents, glidants, and anti-adhesives (e.g., magnesiumstearate, zinc stearate, stearic acid, silicas, hydrogenated vegetableoils, or talc). Compositions for oral use may also be provided aschewable tablets, or as hard gelatin capsules wherein the activeingredient is mixed with an inert solid diluent, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

The compositions herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

VII. Biophysical and Biochemical Characterization

Binding of an anti-PD-L1 Adnectin described herein to PD-L1 may beassessed in terms of equilibrium constants (e.g., dissociation, K_(D))and in terms of kinetic constants (e.g., on-rate constant, k_(on) andoff-rate constant, k_(off)). An Adnectin will generally bind to a targetmolecule with a K of less than 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200pM, or 100 pM, although higher K_(D) values may be tolerated where thek_(off) is sufficiently low or the k_(on), is sufficiently high.

In Vitro Assays for Binding Affinity

An Anti-PD-L1 Adnectin that binds to and antagonizes PD-1 can beidentified using various in vitro assays. In certain embodiments, theassays are high-throughput assays that allow for screening multiplecandidate Adnectins simultaneously.

Exemplary assays for determining the binding affinity of an anti-PD-L1Adnectin includes, but is not limited to, solution phase methods such asthe kinetic exclusion assay (KinExA) (Blake et al., JBC 1996;271:27677-85; Drake et al., Anal Biochem 2004; 328:35-43), surfaceplasmon resonance (SPR) with the Biacore system (Uppsala, Sweden)(Welford et al., Opt. Quant. Elect 1991; 23:1; Morton and Myszka,Methods in Enzymology 1998; 295:268) and homogeneous time resolvedfluorescence (HTRF) assays (Newton et al., J Biomol Screen 2008;13:674-82; Patel et al., Assay Drug Dev Technol 2008; 6:55-68).

In certain embodiments, biomolecular interactions can be monitored inreal time with the Biacore system, which uses SPR to detect changes inthe resonance angle of light at the surface of a thin gold film on aglass support due to changes in the refractive index of the surface upto 300 nm away. Biacore analysis generates association rate constants,dissociation rate constants, equilibrium dissociation constants, andaffinity constants. Binding affinity is obtained by assessing theassociation and dissociation rate constants using a Biacore surfaceplasmon resonance system (Biacore, Inc.). A biosensor chip is activatedfor covalent coupling of the target. The target is then diluted andinjected over the chip to obtain a signal in response units ofimmobilized material. Since the signal in resonance units (RU) isproportional to the mass of immobilized material, this represents arange of immobilized target densities on the matrix, Association anddissociation data are fit simultaneously in a global analysis to solvethe net rate expression for a 1:1 bimolecular interaction, yielding bestfit values for k_(on), k_(off) and R_(max) (maximal response atsaturation). Equilibrium dissociation constants for binding, K_(D)'s arecalculated from SPR measurements as k_(off)/k_(on).

In some embodiments, the anti-PD-L1 Adnectins described herein exhibit aK_(D) in the SPR affinity assay described in Example 2 of 500 nM orless, 400 nM or less, 300 nM or less, 200 nM or less, 150 nM or less,100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM orless, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 15 nMor less, 10 nM or less, 5 nM or less, or 1 nM or less.

It should be understood that the assays described herein above areexemplary, and that any method known in the art for determining thebinding affinity between proteins (e.g., fluorescence based-transfer(FRET), enzyme-linked immunosorbent assay, and competitive bindingassays (e.g., radioimmunoassays)) can be used to assess the bindingaffinities of the anti-PD-L1 Adnectins described herein.

IX. In Vivo Imaging with Anti-PD-L1 Adnectins

Imaging Agents

The anti-PD-L1 Adnectins described herein also are useful in a varietyof diagnostic and imaging applications. In certain embodiments, ananti-PD-L1 Adnectin is labelled with a moiety that is detectable in vivoand such labelled Adnectins may be used as in vivo imaging agents, e.g.,for whole body imaging. For example, in one embodiment, a method fordetecting a PD-L1 positive tumor in a subject comprises administering tothe subject an anti-PD-L1 Adnectin linked to a detectable label, andfollowing an appropriate time, detecting the label in the subject.

An anti-PD-L1 Adnectin imaging agent may be used to diagnose a disorderor disease associated with increased levels of PD-L1, for example, acancer in which a tumor selectively overexpresses PD-L1. In a similarmanner, an anti-PD-L1 Adnectin can be used to monitor PD-L1 levels in asubject, e.g., a subject that is being treated to reduce PD-L1 levelsand/or PD-L1 positive cells (e.g., tumor cells). The anti-PD-L1Adnectins may be used with or without modification, and may be labeledby covalent or non-covalent attachment of a detectable moiety.

Detectable moieties that may be used include radioactive agents, suchas: radioactive heavy metals such as iron chelates, radioactive chelatesof gadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ¹⁸F, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ¹²⁴I, ⁸⁶Y, ⁸⁹Zr, ⁶⁶Ga,⁶⁷Ga, ⁶⁸Ga, ⁴⁴Sc, ⁴⁷SC, ¹¹C, ¹¹¹In, ^(114m) In, ¹¹⁴In, ¹²⁵I, ¹²⁴I, ¹³¹I,¹²³I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr,¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁵Ac, or¹⁵³Sm.

In certain embodiments, the radioactive agent is conjugated to theAdnectin at one or more amino acid residues. In certain embodiments, oneor more, such as two or more, three or more, four or more, or a greaternumber of radionuclides can be present in the labelled probe. In certainembodiments, the radionuclide is attached directly to the Adnectin by achelating agent (e.g., see U.S. Pat. No. 8,808,665). In certainembodiments, the radionuclide is present in a prosthetic groupconjugated to the Adnectin by a bifunctional chelator or conjugating(BFC) moiety. In certain embodiments, the radionuclide chelating agentand/or conjugating moiety is DFO, DOTA and its derivatives (CB-DO2A,3p-C-DEPA, TCMC, Oxo-DO3A), DBCO, TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P,MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTA, NETA,TACN-TM, DTPA, 1B4M-DTPA, CHX-A″-DTPA, TRAP (PRP9), NOPO, AAZTA andderivatives (DATA), H₂dedpa, H₄octapa, H₂azapa, H₅decapa, H₆phospa,HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA basedchelating agents, and close analogs and derivatives thereof.

In certain embodiments, the radionuclide chelating or conjugating (BFC)moiety is maleamide-NODAGA or maleamide-DBCO, which can be attachedcovalently to a polypeptide via cysteine residues near the C-terminus ofthe polypeptide. In certain embodiments, an anti-PD-L1 Adnectin ismodified at its C-terminus by the addition of a cysteine. For example,PxCy may be linked C-terminal to the amino acid residues NYRT, wherein Pis proline, C is cysteine, and x and y are integers that are at least 1.Exemplary anti-PD-L1 Adnectins having the amino acid residues PC attheir C-terminus are set forth in the Examples. Maleimide-NODAGA ormaleimide-DBCO can be reacted with the cysteine, to yieldAdnectin-NODAGA or Adnectin-DBCO, respectively.

In certain embodiments, the radionuclide chelating agent is DFO, whichcan be attached, e.g., at random surface lysines.

In certain embodiments, the chelator for ⁶⁴Cu is DOTA, NOTA, EDTA, Df,DTPA, or TETA. Suitable combinations of chelating agents andradionuclides are extensively reviewed in Price et al., Chem Soc Rev2014; 43:260-90.

In certain embodiments, an anti-PD-L1 Adnectin is labelled with the PETtracer ¹⁸F. ¹⁸F is an attractive PET radionuclide with a 1.8 hourradioactive half life, which provides a same day imaging tool, where thePET radionuclide better matches the Adnectin's biological half-life,resulting in excellent images with less radiation exposure to thepatient. A PD-L1 Adnectin may be labelled with a prosthetic group, suchas[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine([¹⁸F]-FFPEGA), as further described in the Examples. As further shownin the Examples, an ¹⁸F-labelled anti-PD-L1 Adnectin specifically andefficiently labelled human PD-L1 positive tumors in mice and PD-L1positive tumors in cynomolgus monkeys. Specific details on the labellingmethod is provided below and in the Examples.

In certain embodiments, a PD-L1 imaging agent is an anti-PD-L1 Adnectinthat is labelled with ⁶⁴Cu, e.g., as described in the Examples. ⁶⁴Cu maybe linked to an Adnectin with a chelating agent, such as NODAGA. Asfurther shown in the Examples, a ⁶⁴Cu-labelled anti-PD-L1 Adnectinspecifically and efficiently labelled human PD-L1 positive tumors inmice and PD-L1 positive tumors in cyno.

Other art-recognized methods for labelling polypeptides withradionuclides such as ⁶⁴Cu and ¹⁸F for synthesizing the anti-PD-L1Adnectin-based imaging agents described herein may also be used. See,e.g., US2014/0271467; Gill et al., Nature Protocols 2011; 6:1718-25;Berndt et al. Nuclear Medicine and Biology 2007; 34:5-15, Inkster etal., Bioorganic & Medicinal Chemistry Letters 2013; 23:3920-6, thecontents of which are herein incorporated by reference in theirentirety.

In certain embodiments, a PD-L1 imaging agent comprises a PEG molecule(e.g., 5 KDa PEG, 6 KDa PEG, 7 KDa PEG, 8 KDa PEG, 9 KDa PEG, or 10 KDaPEG) to increase the blood PK of the imaging agent by small incrementsto enhance the imaging contrast or increase avidity of the anti-PD-L1Adnectin based imaging agent.

Administration and Imaging

In certain embodiments, the labeled anti-PD-L1 Adnectins can be used toimage PD-L1-positive cells or tissues, e.g., PD-L1 expressing tumors.For example, the labeled anti-PD-L1 Adnectin is administered to asubject in an amount sufficient to uptake the labeled Adnectin into thetissue of interest (e.g., the PD-L1-expressing tumor). The subject isthen imaged using an imaging system such as PET for an amount of timeappropriate for the particular radionuclide being used. The labeledanti-PD-L1 Adnectin-bound PD-L1-expressing cells or tissues, e.g.,PD-L1-expressing tumors, are then detected by the imaging system.

PET imaging with a PD-L1 imaging agent may be used to qualitatively orquantitatively detect PD-L1. A PD-L1 imaging agent may be used as abiomarker, and the presence or absence of a PD-L1 positive signal in asubject may be indicative that, e.g., the subject would be responsive toa given therapy, e.g., a cancer therapy, or that the subject isresponding or not to a therapy.

In certain embodiments, the progression or regression of disease (e.g.,tumor) can be imaged as a function of time or treatment. For instance,the size of the tumor can be monitored in a subject undergoing cancertherapy (e.g., chemotherapy, radiotherapy) and the extent of regressionof the tumor can be monitored in real-time based on detection of thelabeled anti-PD-L1 Adnectin.

The amount effective to result in uptake of the imaging agent (e.g.,¹⁸F-Adnectin imaging agent, ⁶⁴Cu-Adnectin imaging agent) into the cellsor tissue of interest (e.g., tumors) may depend upon a variety offactors, including for example, the age, body weight, general health,sex, and diet of the host; the time of administration; the route ofadministration; the rate of excretion of the specific probe employed;the duration of the treatment; the existence of other drugs used incombination or coincidental with the specific composition employed; andother factors.

In certain embodiments, imaging of tissues expressing PD-L1 is effectedbefore, during, and after administration of the labeled anti-PD-L1Adnectin.

In certain embodiments, the subject is a mammal, for example, a human,dog, cat, ape, monkey, rat, or mouse.

In certain embodiments, the anti-PD-L1 Adnectins described herein areuseful for PET imaging of lungs, heart, kidneys, liver, and skin, andother organs, or tumors associated with these organs which expressPD-L1.

In certain embodiments, the anti-PD-L1 imaging agents provide a contrastof at least 50%, 75%, 2, 3, 4, 5 or more. The Examples show that allanti-PD-L1 Adnectins that were used provided a PET contrast of 2 ormore, and that the affinity of the Adnectins was not important.

When used for imaging (e.g., PET) with short half-life radionuclides(e.g., ¹⁸F), the radiolabeled anti-PD-L1 Adnectins are preferablyadministered intravenously. Other routes of administration are alsosuitable and depend on the half-life of the radionuclides used.

In certain embodiments, the anti-PD-L1 imaging agents described hereinare used to detect PD-L1 positive cells in a subject by administering tothe subject an anti-PD-L1 imaging agent disclosed herein, and detectingthe imaging agent, the detected imaging agent defining the location ofthe PD-L1 positive cells in the subject. In certain embodiments, theimaging agent is detected by positron emission tomography.

In certain embodiments, the anti-PD-L1 imaging agents described hereinare used to detect PD-L1 expressing tumors in a subject by administeringto the subject an anti-PD-L1 imaging agent disclosed herein, anddetecting the imaging agent, the detected imaging agent defining thelocation of the tumor in the subject. In certain embodiments, theimaging agent is detected by positron emission tomography.

In certain embodiments, an image of an anti-PD-L1 imaging agentdescribed herein is obtained by administering the imaging agent to asubject and imaging in vivo the distribution of the imaging agent bypositron emission tomography.

Disclosed herein are methods of obtaining a quantitative image oftissues or cells expressing PD-L1, the method comprising contacting thecells or tissue with an anti-PD-L1 imaging agent described herein anddetecting or quantifying the tissue expressing PD-L1 using positronemission tomography.

Also disclosed herein are methods of detecting a PD-L1-expressing tumorcomprising administering an imaging-effective amount of an anti-PD-L1imaging agent described herein to a subject having a PD-L1-expressingtumor, and detecting the radioactive emissions of said imaging agent inthe tumor using positron emission tomography, wherein the radioactiveemissions are detected in the tumor.

Also disclosed herein are methods of diagnosing the presence of aPD-L1-expressing tumor in a subject, the method comprising

(a) administering to a subject in need thereof an anti-PD-L1 imagingagent described herein; and

(b) obtaining an radio-image of at least a portion of the subject todetect the presence or absence of the imaging agent;

wherein the presence and location of the imaging agent above backgroundis indicative of the presence and location of the disease.

Also disclosed herein are methods of monitoring the progress of ananti-tumor therapy against PD-L1-expressing tumors in a subject, themethod comprising

(a) administering to a subject in need thereof an anti-PD-L1 imagingagent described herein at a first time point and obtaining an image ofat least a portion of the subject to determine the size of the tumor;

(b) administering an anti-tumor therapy to the subject;

(c) administering to the subject the imaging agent at one or moresubsequent time points and obtaining an image of at least a portion ofthe subject at each time point;

wherein the dimension and location of the tumor at each time point isindicative of the progress of the disease.

PET Imaging

Typically, for PET imaging purposes it is desirable to provide therecipient with a dosage of Adnectin that is in the range of from about 1mg to 200 mg as a single intravenous infusion, although a lower orhigher dosage also may be administered as circumstances dictate.Typically, it is desirable to provide the recipient with a dosage thatis in the range of from about 1 mg to 10 mg per square meter of bodysurface area of the protein or peptide for the typical adult, although alower or higher dosage also may be administered as circumstancesdictate. Examples of dosages proteins or peptides that may beadministered to a human subject for imaging purposes are about 1 to 200mg, about 1 to 70 mg, about 1 to 20 mg, and about 1 to 10 mg, althoughhigher or lower doses may be used.

In certain embodiments, administration occurs in an amount ofradiolabeled Adnectin of between about 0.005 μg/kg of body weight toabout 50 μg/kg of body weight per day, usually between 0.02 μg/kg ofbody weight to about 3 μg/kg of body weight. The mass associated with aPET tracer is in the form of the natural isotope (e.g., ¹⁹F for a ¹⁸FPET tracer). A particular analytical dosage for the instant compositionincludes from about 0.5 μg to about 100 μg of a radiolabeled protein.The dosage will usually be from about 1 μg to about 50 μg of aradiolabeled protein.

Dosage regimens are adjusted to provide the optimum detectable amountfor obtaining a clear image of the tissue or cells which uptake theradiolabeled Adnectin. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects towhich the radiolabeled Adnectin is to be administered. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the targeting portion ofthe radiolabeled Adnectin; (b) the tissue or cells to be targeted; (c)the limitations inherent in the imaging technology used.

For administration of the radiolabeled Adnectin, the dosage used willdepend upon the disease type, targeting compound used, the age, physicalcondition, and gender of the subject, the degree of the disease, thesite to be examined, and others. In particular, sufficient care has tobe taken about exposure doses to a subject. Preferably, a saturatingdose of radiolabel (e.g., ¹⁸F or ⁶⁴Cu) is administered to the patient.For example, the amount of radioactivity of F-labeled Adnectin usuallyranges from 3.7 megabecquerels to 3.7 gigabecquerels, and preferablyfrom 18 megabecquerels to 740 megabecquerels. Alternatively, the dosagemay be measured by millicuries, for example. In some embodiments, theamount of ¹⁸F imaging administered for imaging studies is 5 to 10 mCi.In some embodiments, an effective amount will be the amount of compoundsufficient to produce emissions in the range of from about 1-5 mCi.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desired uptakeof the radiolabeled Adnectin in the cells or tissues of a particularpatient, composition, and mode of administration, without being toxic tothe patient. It will be understood, however, that the total daily usageof the radiolabeled Adnectin of the present disclosure will be decidedby the attending physician or other attending professional within thescope of sound medical judgment. The specific effective dose level forany particular subject will depend upon a variety of factors, includingfor example, the activity of the specific composition employed; thespecific composition employed; the age, body weight, general health,sex, and diet of the host; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; other drugs, compounds and/or materialsused in combination with the particular compositions employed, the age,sex, weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.In certain embodiments, the amount of radiolabeled Adnectin administeredinto a human subject required for imaging will be determined by theprescribing physician with the dosage generally varying according to thequantity of emission from the radionuclide.

A composition described herein can be administered via one or moreroutes of administration using one or more of a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for radiolabeled Adnectindescribed herein include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, a radiolabeled Adnectin described herein can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

In certain embodiments, the radiolabeled Adnectin described herein canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds.Agents may cross the BBB by formulating them, for example, in liposomes.For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos.4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one ormore moieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134);p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994).

Exemplary PET Procedure

The following illustrative procedure may be utilized when performing PETimaging studies on patients in the clinic. A 20 G two-inch venouscatheter is inserted into the contralateral ulnar vein for radiotraceradministration. Administration of the PET tracer is often timed tocoincide with time of maximum (T max) or minimum (T min) of theanti-PD-L1 Adnectin concentration in the blood.

The patient is positioned in the PET camera and a tracer dose of the PETtracer of radiolabeled anti-PD-L1 Adnectin such as [¹⁸F]-ADX_5417_E01(<20 mCi) is administered via i.v. catheter. Either arterial or venousblood samples are taken at 15 appropriate time intervals throughout thePET scan in order to analyze and quantitate the fraction ofunmetabolized PET tracer of [¹⁸F]-ADX_5417_E01 in plasma. Images areacquired for up to 120 min. Within ten minutes of the injection ofradiotracer and at the end of the imaging session, 1 ml blood samplesare obtained for determining the plasma concentration of any unlabeledanti-PD-L1 Adnectin which may have been administered before the PETtracer.

Tomographic images are obtained through image reconstruction. Fordetermining the distribution of radiotracer, regions of interest (ROIs)are drawn on the reconstructed image including, but not limited to, thelungs, liver, heart, kidney, skin, or other organs and tissue (e.g.,cancer tissue). Radiotracer uptakes over time in these regions are usedto generate time activity curves (TAC) obtained in the absence of anyintervention or in the presence of the unlabeled anti-PD-L1 Adnectin atthe various dosing paradigms examined. Data are expressed asradioactivity per unit time per unit volume (μci/cc/mCi injected dose).

X. Detection of PD-L1 with Anti-PD-L1 Adnectins

In addition to detecting PD-L1 in vivo, anti-PDL1 Adnectins, such asthose described herein, may be used for detecting a target molecule in asample. A method may comprise contacting the sample with an anti-PD-L1Adnectins described herein, wherein said contacting is carried out underconditions that allow anti-PD-L1 Adnectin-target complex formation; anddetecting said complex, thereby detecting said target in said sample.Detection may be carried out using any art-recognized technique, suchas, e.g., radiography, immunological assay, fluorescence detection, massspectroscopy, or surface plasmon resonance. The sample may be from ahuman or other mammal. For diagnostic purposes, appropriate agents aredetectable labels that include radioisotopes, for whole body imaging,and radioisotopes, enzymes, fluorescent labels and other suitableantibody tags for sample testing.

The detectable labels can be any of the various types used currently inthe field of in vitro diagnostics, including particulate labelsincluding metal sols such as colloidal gold, isotopes such as I¹²⁵ orTc⁹⁹ presented for instance with a peptidic chelating agent of the N₂S₂,N₃S or N₄ type, chromophores including fluorescent markers, biotin,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. A biotinylated antibody would then bedetectable by avidin or streptavidin binding. Suitable enzyme labelsinclude horseradish peroxidase, alkaline phosphatase and the like. Forinstance, the label can be the enzyme alkaline phosphatase, detected bymeasuring the presence or formation of chemiluminescence followingconversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxy phenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo {3.3.1.1 3,7}decan}-4-yl) phenylphosphate (CSPD), as well as CDP and CDP-Star® or other luminescentsubstrates well-known to those in the art, for example the chelates ofsuitable lanthanides such as Terbium(III) and Europium(III). Otherlabels include those set forth above in the imaging section. Thedetection means is determined by the chosen label. Appearance of thelabel or its reaction products can be achieved using the naked eye, inthe case where the label is particulate and accumulates at appropriatelevels, or using instruments such as a spectrophotometer, a luminometer,a fluorimeter, and the like, all in accordance with standard practice.

In certain embodiments, conjugation methods result in linkages which aresubstantially (or nearly) non-immunogenic, e.g., peptide- (i.e. amide-),sulfide-, (sterically hindered), disulfide-, hydrazone-, and etherlinkages. These linkages are nearly non-immunogenic and show reasonablestability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol.13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and Adnectin,different conjugation strategies can be employed. In case the moiety isnaturally occurring or recombinant polypeptide of between 50 to 500amino acids, there are standard procedures in textbooks describing thechemistry for synthesis of protein conjugates, which can be easilyfollowed by the skilled artisan (see e.g. Hackenberger, C. P. R., andSchwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). Inone embodiment the reaction of a maleinimido moiety with a cysteineresidue within the Adnectin or the moiety is used. Alternatively,coupling to the C-terminal end of the Adnectin is performed. C-terminalmodification of a protein can be performed as described in, e.g.,Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371). When themoiety is a peptide or polypeptide, the Adnectin and moiety can be fusedby standard genetic fusion, optionally with a linker disclosed herein.

In general, site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136.Protease-catalyzed formation of C—N bonds is described at Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403.

Site specific reaction and covalent coupling can also be achieved by theselective reaction of terminal amino acids with appropriate modifyingreagents. The reactivity of an N-terminal cysteine with benzonitrils(see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662)can be used to achieve a site-specific covalent coupling. Nativechemical ligation can also rely on C-terminal cysteine residues (Taylor,E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22(Protein Engineering), 65-96). EP 1 074 563 describes a conjugationmethod which is based on the faster reaction of a cysteine within astretch of negatively charged amino acids than a cysteine located in astretch of positively charged amino acids.

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non-labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from un-bound components,like a labeled monovalent binder.

XI. Synthesis of ¹⁸F-Labeled Anti-PD-L1 Adnectins

¹⁸F-labeled anti-PD-L1 Adnectins may be synthesized by first preparingan ¹⁸F radiolabeled prosthetic group, linking an Adnectin to abifunctional chelating agent, and then combining these two reagents(see, e.g., FIG. 9).

¹⁸F Radiolabeled Prosthetic Groups

In one aspect, provided herein is an ¹⁸F-radiolabeled compoundcontaining a prosthetic group for use in a bioorthogonal reactioninvolving 1,3-dipolar cycloaddition between an azide and a cyclooctynewhich proceeds selectively under water tolerant conditions. The¹⁸F-radiolabeled prosthetic groups disclosed herein are soluble in 100%aqueous, and there is no need for an organic phase to link theprosthetic groups to the anti-PD-L1 Adnectins disclosed herein. Thisfeature is particularly advantageous as there is no need for an organicphase to link the prosthetic group to the anti-PD-L1 Adnectins, whichcannot withstand even small amounts of organic solvents, givendegradation and aggregation issues.

Additionally, unlike aliphatic prosthetic groups, the ¹⁸F fluorinationreaction can be monitored with UV, and the ¹⁸F-radiolabeled prostheticgroups described herein are not volatile. Moreover, the ¹⁸F-radiolabeledprosthetic groups can be incorporated into the anti-PD-L1 Adnectinsusing a copper free click chemistry, e.g., as described in the Examples,thus avoiding the stability issues observed in some biologics whencopper mediated click chemistry is used.

In one aspect, provided herein is a PEGylated ¹⁸F-pyridine covalentlybound to an azide with the following structure,

wherein x is an integer from 1 to 8. In certain embodiments, x is aninteger from 2 to 6. In some embodiments x is an integer from 3 to 5. Incertain embodiments, x is 4. In certain embodiments, ¹⁸F is attached tothe pyridine ortho to the N atom. In certain embodiments, the[O(CH₂)₂]_(x) moiety is present in the 1-3 configuration relative to thenitrogen on the pyridine ring. In certain embodiments, the [O(CH₂)₂]_(x)moiety is present in the 1-2 configuration relative to the nitrogen onthe pyridine ring. In certain embodiments, the [O(CH₂)₂]_(x) moiety ispresent in the 1-4 configuration relative to the nitrogen on thepyridine ring.

In certain embodiments, the ¹⁸F-radiolabeled compound has the structure

wherein x is an integer from 1 to 8. In certain embodiments, x is aninteger from 2 to 6. In some embodiments x is an integer from 3 to 5. Incertain embodiments, x is 4.

In certain embodiments, the ¹⁸F-radiolabeled compound has the structure

wherein x is an integer from 1 to 8. In certain embodiments, x is aninteger from 2 to 6. In certain embodiments x is an integer from 3 to 5.In certain embodiments, x is 4.

In certain embodiments, the ¹⁸F-radiolabeled compound is[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine(¹⁸F-FPPEGA) and has the structure

In certain embodiments, the ¹⁸F-radiolabeled prosthetic group maycontain additional groups on the pyridine ring which do not interferewith the fluorination reaction. In certain embodiments, additions to thepyridine ring include C₁₋₆ alkyl groups, for example methyl, ethyl andpropyl.

In certain embodiments, the ¹⁸F-radiolabeled prosthetic group is a fusedring system with the following structure:

wherein “OPEG” is [O(CH₂)₂]_(x), and x is an integer from 1 to 8. Incertain embodiments, x is an integer from 2 to 6. In certain embodimentsx is an integer from 3 to 5. In certain embodiments, x is 4.

The ¹⁸F-radiolabeled prosthetic groups described herein may be producedusing chemical reactions described in the Examples herein.

Also provided herein is a method of preparing a PEGylated ¹⁸F-pyridinecovalently bound to an azide with the following structure,

wherein x is an integer from 1 to 8, the method comprising the steps of

(a) providing a solution of a compound a with the following structure:

wherein x is an integer from 1 to 8, and R is NO₂, Br, F or

and is ortho to the N atom of the pyridine ring;

(b) providing a mixture of ¹⁸F in ¹⁸O water,4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and a weakbase;

c) drying the mixture from step b) to form a solid; and

d) reacting the solution from step a) with the solid from step c) toform the ¹⁸F-labeled compound.

In certain embodiments, the method produces a ¹⁸F-pyridine prostheticgroup with the following structure b

(where ¹⁸F is ortho to the N atom), and includes the steps of

a) providing a solution of the compound of the structure

(where X is ortho to the N atom) where X is NO₂, Br or

b) providing a mixture of ¹⁸F in ¹⁸O water,4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and weakbase, such as K₂CO₃;

c) drying the mixture from step b) to form a solid; and

d) reacting the solution from step a) with the solid from step c) toform the ¹⁸F-labeled compound.

In certain embodiments, the method further comprises the step ofproducing a compound with the following structure a

according to the Scheme I shown below:

In certain embodiments, the method comprises producing ¹⁸F-pyridineprosthetic group is[F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine(¹⁸F-FPPEGA), e, from d according to the following reaction conditions:

In some aspects, provided herein are F-radiolabeled probes or agentswith the following structure,

wherein the Protein is a PD-L1 Adnectin and x is an integer from 1 to 8.In certain embodiments, x is an integer from 2 to 6. In certainembodiments x is an integer from 3 to 5. In some embodiments, x is 4.

BFC

Bifunctional chelating or conjugating (BFC) moieties which can be usedin the ¹⁸F-radiolabled compositions disclosed herein are commerciallyavailable (e.g., Sigma Aldrich; Click Chemistry Tools), or may besynthesized according to well-known chemical reactions.

In certain embodiments, the BFC is selected from cyclooctyne basedchelating agents (e.g., DBCO, DIBO), DFO, DOTA and its derivatives(CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A), TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P,MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTA, NETA,TACN-TM, DTPA, 1B4M-DTPA, CHX-A″-DTPA, TRAP (PRP9), NOPO, AAZTA andderivatives (DATA), H₂dedpa, H₄octapa, H₂azapa, H₅decapa, H₆phospa,HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA basedchelating agents, and close analogs and derivatives thereof. Suitablecombinations of chelating agents and radionuclides are extensivelydescribed in Price et al., Chem Soc Rev 2014; 43:260-90.

In certain embodiments, the BFC is a cyclooctyne comprising a reactivegroup that forms a covalent bond with an amine, carboxyl, carbonyl orthiol functional group on the targeting protein or peptide. Reactivegroups on the cyclooctyne include esters, acids, hydroxyl groups,aminooxy groups, malaiemides, α-halogenketones and α-halogenacetamides.

In certain embodiments, the BFC is a cyclooctyne is dibenzocyclooctyne(DIBO), biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC)and dibenzocyclooctyne (DBCO). In certain embodiments, the cyclootyne isDBCO.

In certain embodiments, the cyclooctyne comprises a hydrophilicpolyethylene glycol (PEG)_(y) spacer arm, wherein y is an integer from 1to 8. In certain embodiments, y is an integer from 2 to 6. In certainembodiments, y is 4 or 5.

In certain embodiments, the BFC is DBCO-PEG4-NHS-Ester orDBCO-Sulfo-NHS-Ester which react specifically and efficiently with aprimary amine (e.g., side chain of lysine residues or aminosilane-coatedsurfaces). In certain embodiments, the BFC is DBCO-PEG4-Acid withterminal carboxylic acid (—COOH) that can be reacted with primary orsecondary amine groups in the presence activators (e.g. EDC) forming astable amide bond. In certain embodiments, the BFC is DBCO-PEG4-Aminewhich reacts with carboxyl groups in the presence of activators (e.g.EDC, or DCC) or with activated esters (e.g. NHS esters) forming stableamide bonds.

In certain embodiments, the BFC is DBCO-PEG4-Maleimide which reacts withsulfhydryl groups on cysteine residues, e.g., at or near the C-terminusof the polypeptide.

In certain embodiments, the polypeptide is modified at its C-terminus bythe addition of a cysteine. For example, P_(m)C_(n) may be linked to theC-terminal amino acid residue of the polypeptide, wherein P is proline,C is cysteine, m is an integer that at least 0 and n is an integer thatis at least 1. Methods for making such modifications are well-known inthe art.

In certain embodiments, the ¹⁸F-radiolabeled probe or agent has thefollowing structure a,

wherein the BFC is conjugated to the protein (e.g., an anti-PD-L1Adnectin) at a cysteine residue.

The ¹⁸F-radiolabeled targeting agents described herein are producedusing bioorthogonal, metal free click chemistry in medium suitable fordirect use in vivo (e.g., saline) according to the procedures describedherein.

XII. Therapeutic Methods Immunotherapy of Cancer Patients Using anAnti-PD-L1 Adnectin

PD-L1 is the primary PD-1 ligand up-regulated within solid tumors, whereit can inhibit cytokine production and the cytolytic activity ofPD-1-positive, tumor-infiltrating CD4⁺ and CD8⁺ T-cells, respectively(Dong et al, 2002; Hino et al, 2010; Taube et al, 2012). Theseproperties make PD-L1 a promising target for cancer immunotherapy. Forexample, clinical trials with anti-PD-L1 immunotherapy, as described inWO2013/173223, which is herein incorporated by reference in itsentirety, demonstrate that mAb blockade of the immune inhibitory ligand,PD-L1, produces both durable tumor regression and prolonged (>24 weeks)disease stabilization in patients with metastatic NSCLC, MEL, RCC andOV, including those with extensive prior therapy. Accordingly, targetingPD-L1, e.g., using the PD-L1 Adnectins, are suitable for eliciting ananti-tumor response.

Based on the clinical data disclosed in WO2013/173223, described hereinare methods for immunotherapy of a subject afflicted with cancer, whichmethod comprises administering to the subject a composition comprising atherapeutically effective amount of a PD-L1 Adnectin described herein.The disclosure also provides a method of inhibiting growth of tumorcells in a subject, comprising administering to the subject a PD-L1Adnectin described herein. In certain embodiments, the subject is ahuman.

Detailed methods for treating a subject with cancer by targeting PD-L1are described in WO2013/173223.

In certain embodiments, the PD-L1 Adnectins described herein, which aresuitable for use in the methods described herein, have one or more ofthe following features: high affinity for human PD-L1 increases T-cellproliferation, increases IL-2 secretion, e.g., from T cells, increasesinterferon-γ production, e.g., from T cells, inhibits the binding ofPD-L1 to PD-1, and reverses the suppressive effect of T regulatory cellson T cell effector cells and/or dendritic cells.

Adnectin Drug Conjugates (Adnectin-DCs)

Adnectins described herein may be conjugated through a cysteine, e.g., aC-terminal cysteine, to a therapeutic agent to form an immunoconjugatesuch as an Adnectin-drug conjugate (“Adnectin-DC”).

In an Adnectin-DC, the Adnectin is conjugated to a drug, with theAdnectin-DC functioning as a targeting agent for directing the Adnectinto a target cell expressing its antigen, such as a cancer cell.Preferably, the antigen is a tumor associated antigen, i.e., one that isuniquely expressed or over-expressed by the cancer cell. Once there, thedrug is released, either inside the target cell or in its vicinity, toact as a therapeutic agent. For a review on the mechanism of action anduse of drug conjugates as used with antibodies, e.g., in cancer therapy,see Schrama et al., Nature Rev. Drug Disc. 2006, 5, 147.

Suitable therapeutic agents for use in drug conjugates includeantimetabolites, alkylating agents, DNA minor groove binders, DNAintercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclearexport inhibitors, proteasome inhibitors, topoisomerase I or IIinhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors,antibiotics, and anti-mitotic agents. In an Adnectin-DC, the Adnectinand therapeutic agent preferably are conjugated via a linker cleavablesuch as a peptidyl, disulfide, or hydrazone linker. More preferably, thelinker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 169), Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The Adnectin-DCscan be prepared according to methods similar to those described in U.S.Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; andWO 08/103693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference. A linker can itself be linked, e.g., covalently linked, e.g.,using maleimide chemistry, to a cysteine of the polypeptide, e.g., acysteine at or near the C-terminus of the polypeptide, e.g., a cysteineof a P_(m)C_(n) moiety that is attached to the C-terminal amino acidresidue of the polypeptide, wherein m is an integer that is at least 0(e.g., 0, 1 or 2) and n is an integer that is at least 1 (e.g., 1 or 2).For example, a linker can be covalently linked to a cysteine, such as acysteine in the C-terminal region of an Adnectin, e.g., a C-terminalcysteine in PC of an Adnectin-DC-P_(m)C_(n), wherein m and n areindependently an integer that is at least one. For example, a linker canbe linked to an Adnectin-DC-PmCn, wherein P is a proline, C is acysteine, and m and n are integers that are at least 1, e.g., 1-3. Incertain embodiments, m is zero, i.e., the cysteine is not preceded by aproline. Ligation to a cysteine can be performed as known in the artusing maleimide chemistry (e.g., Taylor, E. Vogel; Imperiali, B, NucleicAcids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).For attaching a linker to a cysteine on an Adnectin, the linker may,e.g. comprise a maleinimido moiety, which moiety then reacts with thecysteine to form a covalent bond. In certain embodiments, the aminoacids surrounding the cysteine are optimized to facilitate the chemicalreaction. For example, a cysteine may be surrounded by negativelycharged amino acid for a faster reaction relative to a cysteine that issurrounded by a stretch of positively charged amino acids (EP 1 074563).

For cancer treatment, the drug preferably is a cytotoxic drug thatcauses death of the targeted cancer cell. Cytotoxic drugs that can beused in Adnectin-DCs include the following types of compounds and theiranalogs and derivatives:

-   -   (a) enediynes such as calicheamicin (see, e.g., Lee et al., J.        Am. Chem. Soc. 1987, 109, 3464 and 3466) and uncialamycin (see,        e.g., Davies et al., WO 2007/038868 A2 (2007) and Chowdari et        al., U.S. Pat. No. 8,709,431 B2 (2012));    -   (b) tubulysins (see, e.g., Domling et al., U.S. Pat. No.        7,778,814 B2 (2010); Cheng et al., U.S. Pat. No. 8,394,922 B2        (2013); and Cong et al., US 2014/0227295 A1;    -   (c) CC-1065 and duocarmycin (see, e.g., Boger, U.S. Pat. No.        6,5458,530 B1 (2003); Sufi et al., U.S. Pat. No. 8,461,117 B2        (2013); and Zhang et al., US 2012/0301490 A1 (2012));    -   (d) epothilones (see, e.g., Vite et al., US 2007/0275904        A1 (2007) and U.S. RE42930 E (2011));    -   (e) auristatins (see, e.g., Senter et al., U.S. Pat. No.        6,844,869 B2 (2005) and Doronina et al., U.S. Pat. No. 7,498,298        B2 (2009));    -   (f) pyrrolobezodiazepine (PBD) dimers (see, e.g., Howard et al.,        US 2013/0059800 A1(2013); US 2013/0028919 A1 (2013); and WO        2013/041606 A1 (2013)); and    -   (g) maytansinoids such as DM1 and DM4 (see, e.g., Chari et al.,        U.S. Pat. No. 5,208,020 (1993) and Amphlett et al., U.S. Pat.        No. 7,374,762 B2 (2008)).

Exemplary Cancers' Treatable by PD-L1 Adnectins

The PD-L1 Adnectins described herein may be suitable for use in thetreatment of a broad range of cancers, including treatment-refractorymetastatic NSCLC, that are generally not considered to beimmune-responsive. Exemplary cancers that may be treated using the PD-L1Adnectins described herein include MEL. (e.g., metastatic malignantmelanoma), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer(OV), gastric cancer (GC), breast cancer (BC), pancreatic carcinoma(PC), and carcinoma of the esophagus. Additionally, the PD-L1 Adnectinsdescribed herein are suitable for use in treating refractory orrecurrent malignancies.

Non-limiting examples of cancers that may be treated using the PD-L1Adnectins, based on the indications of very broad applicability ofanti-PD-L1 immunotherapy disclosed in WO2013/173223, include bonecancer, skin cancer, cancer of the head or neck, breast cancer, lungcancer, cutaneous or intraocular malignant melanoma, renal cancer,uterine cancer, castration-resistant prostate cancer, colon cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, carcinomas of the ovary, gastrointestinal tractand breast, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma, multiplemyeloma, environmentally induced cancers including those induced byasbestos, metastatic cancers, and any combinations of said cancers. ThePD-L1 Adnectins are also applicable to the treatment of metastaticcancers.

Combination Therapy with PD-L1 Adnectins

In certain embodiments, the PD-L1 Adnectins described herein may becombined with an immunogenic agent, for example a preparation ofcancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), antigen-presentingcells such as dendritic cells bearing tumor-associated antigens, andcells transfected with genes encoding immune stimulating cytokines (Heet ah, 2004). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-², MARTI and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF. PD-L1 blockade may also be effectivelycombined with standard cancer treatments, including chemotherapeuticregimes, radiation, surgery, hormone deprivation and angiogenesisinhibitors, as well as another immunotherapeutic antibody (e.g., ananti-PD-1, anti-CTLA-4, and anti-LAG-3 Ab).

Medicaments and Uses of Anti-PD-L1 Adnectins

anti-PD-L1 Adnectin described herein may be used for the preparation ofa medicament for inhibiting signaling from the PD-1/PD-L1 pathway so asto thereby potentiate an endogenous immune response in a subjectafflicted with cancer. This disclosure also provides the use of anyanti-PD-L1 Adnectin described herein for the preparation of a medicamentfor immunotherapy of a subject afflicted with cancer. The disclosureprovides medical uses of any anti-PD-L1 Adnectin described hereincorresponding to all the embodiments of the methods of treatmentemploying a PD-L1 Adnectin described herein.

Also described herein are anti-PD-L1 Adnectins for use in treating asubject afflicted with cancer comprising potentiating an endogenousimmune response in a subject afflicted with cancer by inhibitingsignaling from the PD-1/PD-L1 pathway. The disclosure further providesanti-PD-L1 Adnectins for use in immunotherapy of a subject afflictedwith cancer comprising disrupting the interaction between PD-1 andPD-L1. These anti-PD-L1 Adnectins may be used in potentiating anendogenous immune response against, or in immunotherapy of, the fullrange of cancers described herein. In certain embodiments, the cancersinclude MEL (e.g., metastatic malignant MEL), RCC, squamous NSCLC,non-squamous NSCLC, CRC, ovarian cancer (OV), gastric cancer (GC),breast cancer (BC), pancreatic carcinoma (PC), and carcinoma of theesophagus.

Infectious Diseases

Also described herein are methods for treating patients that have beenexposed to particular toxins or pathogens. For example, in certainaspects, this disclosure provides a method of treating an infectiousdisease in a subject comprising administering to the subject ananti-PD-L1 Adnectin described herein such that the subject is treatedfor the infectious disease.

Similar to its application to tumors as discussed above,Adnectin-mediated PD-L1 blockade can be used alone, or as an adjuvant,in combination with vaccines, to potentiate an immune response topathogens, toxins, and/or self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, and C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. PD-L1 blockade is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of an infection, Novel epitopes on these antigens are recognizedas foreign at the time of anti-PD-L1 Adnectin administration, thusprovoking a strong T cell response that is not dampened by negativesignals through the PD-1/PD-L1 pathway.

In the above methods, PD-L1 blockade can be combined with other forms ofimmunotherapy known in the art, such as cytokine treatment (e.g.,administration of interferons, GM-CSF, G-CSF or IL-2).

XIII. Kits and Articles of Manufacture

The anti-PD-L1 Adnectins described herein can be provided in a kit, apackaged combination of reagents in predetermined amounts withinstructions for use in the therapeutic or diagnostic methods describedherein.

For example, in certain embodiments, an article of manufacturecontaining materials useful for the treatment or prevention of thedisorders or conditions described herein, or for use in the methods ofdetection described herein, are provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer may hold a composition described herein for in vivo imaging,and may have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is ananti-PD-L1 Adnectin described herein. The article of manufacture mayfurther comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

In certain embodiments, a kit comprises one or more reagents necessaryfor forming an ¹⁸F labelled anti-PD-L1 Adnectin in vivo imaging agent,such as a PD-L1 Adnectin-PEG4-DBCO-¹⁸F, as further described herein. Forexample, a kit may comprise a first vial comprising anti-PD-L1Adnectin-PEG-4-DBCO and a second vial comprising [¹⁸F]FPPEGA. A kit maycomprise a first vial comprising anti-PD-L1 Adnectin-PEG-4-DBCO, asecond vial comprising 4-PEG-tosyl-azide and a third vial comprising ¹⁸Fin O¹⁸ water. The kits may further comprise vials, solutions andoptionally additional reagents necessary for the manufacture of PD-L1Adnectin-PEG4-DBCO-¹⁸F. Similarly, kits may comprise the reagentsnecessary for forming a ⁶⁴Cu labelled anti-PD-L1 Adnectin, such as thereagents described herein.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

EXAMPLES Example 1: Identification of PD-L1 Binding Adnectins

Anti-PD-L1 Adnectins were isolated from an Adnectin library screenedwith a human PD-L1 protein, or were affinity matured by PROfusion fromclones identified in the library. The full length sequences, coresequences, BC, DE, and FG loop sequences of these Adnectins, as well asvariants with a “PC” modified C-terminus, are presented in FIG. 1 andTable 3.

For example, the high-affinity, anti-PD-L1 Adnectin, ADX_5322_A02(“A02”), was obtained by affinity-maturing the ATI-964 Adnectin. Thegene encoding ATI-964 was re-diversified by introducing a small fractionof non-wild-type nucleotides at each nucleotide position that encoded aresidue in loop BC, DE, or FG. The resulting library of Adnectinsequences related to ATI-964 was then subjected to in vitro selection byPROfusion (mRNA display) for binding to human PD-L1 underhigh-stringency conditions. The clones enriched after completedselection were sequenced, expressed in HTPP format, and screened fortheir ability to bind PD-L1 and for their fraction of monomericity. Theclone with the best combination of affinity for PD-L1 and robustbiophysical properties was mutated to include a C-terminal Cysteine,first with the C-terminal sequence NYRTPCH6 (the form identified asADX_5322_A02), and later with the C-terminal sequence NYRTPC.

The same process was followed to affinity-mature ATI-967, resulting inthe Adnectin ADX_5417_E01. Similarly, affinity matured ATI_1760_C02,ATI_1760_E01 (“E01”) and ATI_1760_F01 were obtained by affinitymaturation of ATI_1422_G05.

Additional anti-human PD-L1 adnectins were isolated. Their sequences areset forth in Table 3.

Expression and Purification of his-Tagged Anti-PD-L1 Adnectins

All DNA constructs contained an N-terminal his tag followed by a TVMVrecognition sequence. The expression plasmids (pET-28 NM vector) for theanti-PD-L1 Adnectins described supra were transformed into BL21(DE3)cells (New England Biolabs). Cells were grown in Overnight ExpressAutoinduction media (Novagen) in 1L shake flasks at 37° C. for 6 hoursfollowed by 20° C. for 16 hours at 220 RPM. Cells were harvested bycentrifugation and suspended in PBS pH 7.2. Cells were lysedmechanically, then clarified by centrifugation. Soluble fractions werebound by gravity feed to Ni-NTA Agarose resin (Qiagen), washed in 20 mMTris+10 mM Imidazole pH 8.0, followed by 20 mM Tris+40 mM Imidazole pH8.0, and eluted with 20 mM Tris+400 mM Imidazole pH 8.0. Nickel eluateswere spiked with TVMV protease at 1:23-fold molar excess of Adnectin.The TVMV-Adnectin eluate mixtures were dialyzed against 20 mM Tris pH8.0 at 4° C. for 16 hours. To separate the TVMV protease and cleaved histag fragments, samples were loaded onto a 10 mL HisTrap FF column (GEHealthcare) and flow through fractions were collected.

Example 2: Biophysical Assessment of PD-L1 Adnectins

The binding properties of ATI-1420D05, ATI-1420D05, ATI-1421E04 andATI-1422G05, ATI_1760_C02, ATI_1760_E01 and ATI_1760_E01 were assessed.

TABLE 1 Binding properties of anti-PD-L1 Adnectins Sequence Name SEQ IDK_(D)(nM) ATI-1420B09 2.5 ATI-1420D05 9.5 AT1-1421E04 5.6

Binding properties of the purified ATI-964, ATI-967, ATI-968,ADX_5322_A02, and ADX_5417_E01 Adnectins to human or cyno PD-L1, asdetermined by Biacore, are shown in Table 2. Cell binding was determinedby measuring binding to human PD-L1 positive cells L2987.

TABLE 2 Biophysical properties of anti-PD-L1 Adnectins MeltingTransition Cell Binding Kinetics Midpoint Binding SEQ K_(D) (M) for(Tm)° C. EC₅₀ Sequence Name ID k_(a) (1/Ms) k_(d) (s) K_(D) (M)cynoPD-L1 N.D. N.D. ATI-964 30 3.6 × 10⁵ 7.7 × 10⁻⁵ 2.1 × 10⁻¹⁰ 1.4 ×10⁻¹⁰ N.D. 4.4 ATI-965 45 4.6 × 10⁵ 3.3 × 10⁻⁴ 7.1 × 10⁻¹⁰   8 × 10⁻¹⁰N.D. 4.9 ATI-966 60 2.5 × 10⁶ 6.9 × 10⁻⁵ 2.8 × 10⁻¹¹ 3.8 × 10⁻¹¹ N.D.2.3 ATI-967 75 2.1 × 10⁶ 6.7 × 10⁻⁵ 3.2 × 10⁻¹¹   6 × 10⁻¹¹ N.D. N.D.ATI-968 15 7.5 × 10⁵ 1.2 × 10⁻⁴ 1.6 × 10⁻¹⁰ 1.1 × 10⁻¹⁰ 60 3.4ADX_5322_A02 91 2.5 × 10⁶ 5.7 × 10⁻⁴ 2.28 × 10⁻¹⁰  N.D. 82 0.43 nM (Cys-Capped) ADX_5417_E01 107 2.0 × 10⁷ 2.6 × 10⁻⁴ 1.3 × 10⁻¹¹ N.D. 73 N.D.

The binding data indicates that the affinity matured anti-human PD-L1adnectins bind to human PD-L1 with affinities that are less than 1 nM oreven less than 0.1 nM. Exemplary inhibition curves are shown in FIG. 12.

Anti-PD-L1 adnectins have the following additional characteristics:

-   -   Inhibiting the binding of human PD-1 to human PD-L1, as        determined by measuring inhibition of binding of human PD-1Fc to        the PD-L1 positive cells L2987 by flow cytometry, and shown,        e.g., for adnectins ATI-964, ATI-965, ATI-966, ATI-967, ATI-968,        A02 and E01;    -   Inhibiting the binding of human CD80 (B7-1) to human PD-L1, as        determined by ELISA. For example, ATI-964 inhibits binding with        an EC50 of 41 pM; ATI-965 inhibits binding with an EC50 of 210        pM; ATI-966 inhibits binding with an EC50 of 28 pM; and ATI-968        inhibits binding with an EC50 of 56 pM;    -   Inhibiting the binding of the anti-PD-L1 antibody 12A4 to human        PD-L1, as determined by ELISA.

Anti-PD-L1 antibodies were also tested in a mixed lymphocyte reaction(MLR): ATI-964, ATI-965, and ATI-968 were active in an MLR, whereasATI-966 and ATI-967 were not active in an MLR.

The following examples relate to the labeling of anti-PD-L1 Adnectinswith ¹⁸F and ⁶⁴Cu.

Example 3: Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl4-methylbenzenesulfonate

A mixture of ((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (5 g, 9.95 mmol) and sodium azide (0.647g, 9.95 mmol) were dissolved in ethanol (50 mL) and the reaction wasrefluxed at 90° C. over a 17 hour period. The solvent was removed usingpartial vacuum and then loaded onto a 40 gram silica cartridge andpurified using flash chromatography (IscoCombiFlash—eluted using alinear gradient method starting from 10% ethyl acetate in hexanes goingto a 90% ethyl acetate in hexanes over a 45 minute period). The pooledfractions were checked by TLC and combined to give2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate as acolorless oil. Due to the reactive nature of the2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonateproduct this material was used “as is” without any furthercharacterizations.

Example 4: Preparation of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine

To a suspension of sodium hydride (0.129 g, 3.21 mmol) in DMF (10 mL) at0° C. was dropwise added a stirring solution of 2-fluoropyridin-3-ol(0.363 g, 3.21 mmol) in DMF (5 mL), then followed by the dropwiseaddition of the solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl4-methylbenzenesulfonate (1.00 g, 2.68 mmol) in DMF (5 mL). Thesuspension was held at 0° C. for 10 min, then brought to ambienttemperature for 1 hour, followed by additional heating at 60° C. for 4hours. The solvent was removed in vacuo. 100 ml of ethyl acetate wasadded followed by 3 separate wash extractions with concentrated brinesolution. The organic layer was dried over sodium sulfate, filtered, andconcentrated. The crude material was purified using flash chromatography(IscoCombiFlash—eluted with 10-50% EtOAc in Hex) to give a colorlessoil. 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine(702 mg, 2.233 mmol, 83% yield) was isolated as a clear oil. 1H NMR (400MHz, CHLOROFORM-d) δ 7.75 (dt, J=4.9, 1.6 Hz, 1H), 7.33 (ddd, J=10.0,8.1, 1.5 Hz, 1H), 7.10 (ddd, J=7.9, 4.9, 0.7 Hz, 1H), 4.30-4.16 (m, 2H),3.95-3.83 (m, 2H), 3.80-3.61 (m, 10H), 3.38 (t, J=5.1 Hz, 2H) 13C NMR(101 MHz, CHLOROFORM-d) δ 142.3, 137.7, 137.5, 123.4, 123.4, 121.7,121.6, 77.3, 76.7, 70.9, 70.7, 70.6, 70.0, 69.4, 69.0, 50.6 19F NMR (400MHz, CHLOROFORM-d) 6-83.55. HRMS (ESI) Theory:C13H20FN4O4+m/z 315.464.found 315.1463.

Example 5: Preparation of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine

Sodium hydride (0.121 g, 3.01 mmol) (60% suspension in oil) wasdissolved in DMF (7.0 mL) and the resulting suspension was cooled to 0°C. A solution of 2-nitropyridin-3-ol (0.384 g, 2.74 mmol) in DMF (1.5mL) was added slowly, followed by the dropwise addition of2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate(1.023 g, 2.74 mmol) in DMF (1.5 mL). The suspension was held at 0° C.for 10 minutes, then brought to ambient temperature for 2 hours followedby heating 60° C. for a 72 hour period. The reaction was quenched with10 ml of DI water, followed by ethyl acetate extraction (3×10 mL).Pooled EtOAc extracts were washed with a concentrated brine solution (10mL), dried over sodium sulfate, filtered, and evaporated under reducedpressure to give a light yellow oil. The crude was purified by flashchromatography. 24 g silica cartridge, 25 mL/min, starting from 10%ethyl acetate in hexanes, followed by a linear change to 50% ethylacetate in hexanes over a 25 minute period. After this time, thegradient was held at this solvent composition for 10 minutes thenchanged to 100% ethyl acetate over a 10 minute period.3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine waseluted between the 30-40 minute portion of the chromatogram and thepooled fractions were evaporated under reduced pressure, then undervacuum for 2 hours to give3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine (687 mg,1.973 mmol, 72.0% yield) as a light yellow oil. ¹H NMR (400 MHz,CHLOROFORM-d) δ 8.11 (dt, J=4.9, 1.6 Hz, 1H), 7.60 (ddd, J=10.0, 8.1,1.5 Hz, 1H), 7.52 (ddd, J=7.9, 4.9, 0.7 Hz, 1H), 4.30-4.16 (m, 2H),3.95-3.83 (m, 2H), 3.80-3.61 (m, 10OH), 3.38 (t, J=5.1 Hz, 2H) 13C NMR(101 MHz, CHLOROFORM-d) δ 147.3, 139.5, 128.4, 124.4. 71.1, 70.7, 70.6,70.0, 69.9, 69.3, 50.7. HRMS (ESI) Theory:C13H20N5O6+m/z 342.1408. found342.1409.

Example 6: Synthesis of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-bromopyridine

To the suspension of sodium hydride (NaH, 25.7 mg, 0.643 mmol) indimethylformamide (DMF, 5 mL) at 0° C. was dropwise added a solution of2-bromopyridin-3-ol (112 mg, 0.643 mmol) in DMF (1 mL), followed by thedropwise addition of the solution of2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (200mg, 0.536 mmol) in DMF (1 mL). The suspension was held at 0° C. for 10minutes, then brought to ambient temperature and held for 1 hour,followed by heating to 60° C. for 4 hours. Upon completion of heating,the solvent of the crude reaction mixture was removed in vacuo. Thecrude reaction was reconstituted in 50 mL of ethyl acetate, washed with2×50 mL of a aqueous brine solution, and the organic layer was driedover magnesium sulfate, filtered, and concentrated in vacuo. The crudereaction was purified using reverse-phase HPLC to give3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-bromopyridine, TFA(112 mg, 0.229 mmol, 42.7% yield) as a light yellow oil. HRMS ESI m/z(M+H), Theory C13H20BrN4O4 375.0664 found 375.0662. ¹H NMR (400 MHz,DMSO-d₆) δ 7.97 (dd, J=4.6, 1.5 Hz, 1H), 7.54 (dd, J=8.2, 1.6 Hz, 1H),7.40 (dd, J=8.1, 4.6 Hz, 1H), 4.24 (dd, J=5.3, 3.9 Hz, 2H), 3.85-3.78(m, 2H), 3.68-3.62 (m, 2H), 3.62-3.52 (m, 8H), 3.42-3.34 (m, 2H).

Example 7: Scheme for Synthesis of Trimethylanilium Compound

Example 8: Synthesis of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine

A mixture of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (160mg, 0.509 mmol), potassium carbonate (K₂CO₃, 84 mg, 0.611 mmol), anddimethylamine (40% in water, 0.097 mL, 0.764 mmol) in dimethylsulfoxide(DMSO, 2.5 mL) were heated in a sealed pressure-proof vessel at 110° C.for 14 hours. Upon completion of heating, the solvent of the crudereaction mixture was removed in vacuo. The crude reaction wasreconstituted in 50 mL of ethyl acetate, washed with 2×50 mL of aaqueous brine solution, and the organic layer was dried over magnesiumsulfate, filtered, and concentrated in vacuo. The crude reaction waspurified using normal-phase chromatography to give3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine(140 mg, 0.413 mmol, 81% yield) as a colorless oil. 1H NMR (400 MHz,CHLOROFORM-d) δ 7.86 (dd, J=4.9, 1.5 Hz, 1H), 7.02 (dd, J=7.8, 1.5 Hz,1H), 6.73 (dd, J=7.8, 4.9 Hz, 1H), 4.20-4.07 (m, 2H), 3.98-3.86 (m, 2H),3.81-3.61 (m, 9H), 3.38 (t, J=5.1 Hz, 2H), 3.13-2.94 (m, 6H), 1.69 (s,2H). HRMS (ESI) Theory:C15H26N5O4+m/z 340.1980. found 340.1979.

Example 9: Synthesis of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium

Methyl trifluoromethanesufonate (0.065 mL, 0.589 mmol) was added to thesolution of3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine(40 mg, 0.118 mmol) in toluene (1.5 mL) in a sealed container under asteady stream of nitrogen. The reaction mixture was stirred at roomtemperature over a 14 hour period. The solvent was removed and theresultant residue was washed with 2×10 ml of ether, azeotropically driedwith 2×1 ml of dichloromethane, and dried under high-pressure vacuumovernight to give3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium,trifluoromethanesulfonate salt in quantitative yield as a thickcolorless oil. LCMS m/z 354.33; ¹H NMR (400 MHz, DMSO-d₆) δ 8.24-8.17(m, 1H), 7.98 (d, J=8.3 Hz, 1H), 7.75 (ddd, J=8.2, 4.6, 3.2 Hz, 1H),4.44 (br. s., 2H), 3.88 (d, J=3.9 Hz, 2H), 3.69-3.45 (m, 21H).

Example 10: Synthesis of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridineusing3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium,trifluoromethanesulfonate salt

An aqueous [¹⁸F]-Fluoride solution (2.0 ml, 33.3 GBq/900 mCi) waspurchased from P.E.T. Net® Pharmaceuticals in West Point PA and directlytransferred to a Sep-Pak light QMA [The Sep-Pak light QMA cartridge waspre-conditioned sequentially with 5 ml of 0.5 M potassium bicarbonate, 5ml of deionized water, and 5 ml of MeCN before use.] Upon completion ofthis transfer, the aqueous [¹⁸F] fluoride was released from the QMASep-Pak by the sequential addition of potassium carbonate (15 mg/ml; 0.1ml) followed by a mixture of potassium carbonate (30 mg/ml, 0.1 ml),4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (15 mg, 0.04mmol) and 1.2 ml of MeCN. The solvent was evaporated under a gentlestream of nitrogen at 90° C. and vacuum. Azeotropic drying was repeatedtwice with 1 ml portions of acetonitrile to generate the anhydrousK.2.2.2/K[¹⁸F]F complex.3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium,trifluoromethanesulfonate salt (2 mg, 5.6 μmol) was dissolved in 500microliters of DMSO and added to the dried cryptand. This solution washeated at 120° C. for 10 minutes. After this time, the crude reactionmixture was diluted with 3 ml of DI water. The entire contents of thecrude reaction mixture was then transferred, loaded, and purified usingreverse phase HPLC under the following conditions: HPLC Column: Luna C18250×10 Solvent A: 0.1% TFA in DI water; solvent B: 0.1% TFA inacetonitrile at a flow rate of 4.6 ml/minute using isocratic method 32%B while the UV was monitored at 280 nm.[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridinewas isolated at the 24 min mark of the chromatogram and collect over a 2minute period. This product was collected into a 100 ml flask thatcontained 10 ml of DI water and the entire contents were delivered to aSep-Pak Vac tC18 6 cc 1g sep pack from Waters. 6.1 GBq/164 mCi of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridinewas isolated from this reaction. This was released from the sep-pakusing 3 ml of ethanol and this solution was reduced with 98° C. heatsource, a gentle stream of nitrogen, and vacuum over a 15 minute perioduntil only a film remained in the vial. The final product wasreconstituted in 100% 1×PBS buffer and was stable in this media for over1 hour at 37° C.

The[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridinemay be used to generate ¹⁸F-labeled biologic products (e.g., ¹⁸F-labeledanti-PD-L1 Adnectins, as described below) by taking advantage of “click”azide-alkyne reaction with the appropriate biologic containing analkynes.

Example 11: Production of ¹⁸F-Radiolabeled Protein Using “ClickChemistry” A. Fluorination of the 4-PEG-Tosyl-Azide Precursor to Form[¹⁸F]-FPPEGA

900 mCi of ¹⁸F in ¹⁸O water (3 ml) activity (purchased from IBAMolecular) was transferred directly into a micro vial (no QMA) thatcontained 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane(2.8 mg, 7.44 μmol) and potassium carbonate (1.7 mg, 0.012 mmol). Anadditional 2.0 ml of acetonitrile was transferred into this crudereaction mixture and the entire mixture was azeotropically dried. Thiswas completed by evaporating the solution using a 98° C. oil bath, andapplying a gentle stream of N₂ and partial vacuum. The solution's volumewas reduced to about 2 ml. An additional 2 ml of acetonitrile was addedand the process was repeated 3 times over a 40 minute period. When thevolume of the liquid was reduced to less than 0.3 ml, a 0.7 ml aliquotof acetonitrile was added and the solution reduced by further azeotropicdistillation until the volume was ˜0.1 ml. An additional 0.9 ml ofacetonitrile was added and this process was completed until a whitesolid was formed. This process took ˜55 minutes. During the finalprocedure, the vial was removed from the oil bath before the solutionhad gone to dryness and the residue in the vial was placed under fullvacuum (no N₂ flow) at room temperature for 20 minutes. Total time fortransfer and drying of [¹⁸F]-FPPEGA cryptand mixture was 65 min.

To the dried [¹⁸F]-FPPEGA cryptand mixture was added3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine (2 mg,5.86 μmol) dissolved in 500 microliters of DMSO and this mixture washeated at 120° C. for 10 minutes. After this time the crude reactionmixture was diluted with 3 ml of DI water and the entire contents werethen transferred and loaded onto the following HPLC column andconditions: HPLC Column: Luna C18 250×10 mm; Solvent A: 0.1% TFA in DIwater; Solvent B: 0.1% TFA in acetonitrile; flow rate 4.6 ml/min;pressure 1820 PSI; isocratic method 32% B; UV—280 nm. The[F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine([¹⁸F]-FPPEGA) product was isolated at the 24 minute mark of thechromatogram and was collect over a 2 minute period. This product wascollected into a 100 ml flask that contained 15 ml of DI water and theentire contents were delivered to a Sep PakVac tC18 6 cc 1g sep pack. PNWAT036795. The [¹⁸F]-FPPEGA was released from the Sep Pak using 2.5 mlof ethanol and this solution was reduced with 98° C. N₂ and vacuum overa 15 minute period until dryness. This compound was dissolved in 0.1 ml1×PBS (phosphate buffered saline). This product was analyzed using aVarian HPLC HPLC Column Luna C18 (2) 4.6×150 mm Solvent A: 0.1% TFA inDI water; Solvent B: 0.1% TFA in acetonitrile; flow rate 1.0 ml/min;gradient method 0 min 90% A 10% B; 15 mins 30% A 70% B; 17 mins 30% A70% B; 18 mins 90% A 10% B; 20 mins 90% A 10% B; UV—280 nm. 220 mCi of[¹⁸F]-FPPEGA was isolated.

B. Preparation of E01-4PEG-DBCO

This Example describes the linking of the E01 anti-PD-L1 Adnectin toPEG4-DBCO.

As maleimide chemistry is used to link the Adnectin to PEG4-DBCO, theE01 Adnectin was first modified by adding a proline followed by acysteine at its C-terminus. The amino acid sequence of this modified E01Adnectin is provided in SEQ ID NO: 104. The cysteine is used to link theAdnectin to PEG4-DBCO.

A 4-fold molar excess of Maleimide-PEG4-DBCO (Click Chemistry Tools) wasdissolved in DMSO and added to the purified modified E01 Adnectin in thepresence of 1 mM TCEP. Final DMSO concentrations did not exceed 5% inthe conjugation mixtures. The conjugation mixture was left at roomtemperature for one hour before mass spec analysis. After MSconfirmation of conjugation, the sample was purified by size-exclusionchromatography using a HiLoad 26/60 Superdex 75 column (GE Healthcare)equilibrated in PBS pH 7.2.

C. Coupling of [¹⁸F]-FPPEGA to Adnectin

A schematic for synthesizing [¹⁸F]-E01-4PEG-DBCO-FPPEGA is shown inFIGS. 2 and 9.

0.2 ml of a 5.4 mg/ml solution of the E01-4PEG-DBCO Adnectin solution(prepared as described in Section B) was incubated with 200 mCi of 0.1ml of [¹⁸F]-FPPEGA (Example 4) in 1×PBS buffer. The solution was gentlymixed by pipetting the crude reaction up and down several times and wasincubated together for 45 minutes at 45° C. or room temperature. Thecontents of this crude reaction mixture were purified using a SECcolumn. Superdex 200 0.5 ml/min 1×PBS buffer and the[¹⁸F]-E01-4PEG-DBCO-FPPEGA product was isolated at the 37 min mark ofthe chromatogram over a 2 minute period.

[¹⁸F]-E01-4PEG-DBCO-FPPEGA was analyzed via SEC with co-injection ofnon-radioactive standard, RP HPLC using a PLRPS column and gelelectrophoresis.

Size Exclusion Chromatography (SEC) was performed with the followingparameters:

Superdex 200 column; Solvent 100% 1×PBS buffer; 0.5 ml/min 280 UV;

Reverse phase HPLC

Column: PLRPS 8 micron 1000 A 4.6×250 mm

Solvent A: 0.1% formic acid in DI water

Solvent B: Acetonitrile

Flow rate: 1 ml/min

Pressure: 1351 PSI

Gradient:

-   -   0 min 90% A 10% B    -   30 min 45% A 55% B    -   32 min 25% A 75% B    -   36 min 25% A 75% B    -   50 min 90% A 10% B

15 mCi [F]-E01-4PEG-DBCO-FPPEGA was isolated with a radiochemical purity(RCP) of >99% via both SEC and RP HPLC calculations, and with a specificactivity of 0.6 mCi/nmol, when the reaction was conducted at 45° C. Whenconducting the reaction at room temperature, 5.72 mCi was obtained.Specific activity of the [¹⁸F]-FPPEGA was 0.512 mCi/nmol and RCP of85.7% 3 hours post the end of its synthesis, when conducting thereaction at 45° C. or at room temperature, respectively. Specificactivity was measured via Nanodrop (see www.nanodrop.com). The productco-eluted with non-radioactive standard on both SEC and PLRPS. Gelelectrophoresis confirmed an ¹⁸F product consistent with an 11 kDamolecular weight standard.

The ¹⁸F-radiolabeled E01-4PEG-DBCO can be used in a variety of in vitroand/or in vivo imaging applications, including diagnostic imaging, basicresearch, and radiotherapeutic applications. Specific examples ofpossible diagnostic imaging and radiotherapeutic applications, includedetermining the location, the relative activity and/or quantifying ofPD-L1 positive tumors, radioimmunoassay of PD-L1 positive tumors, andautoradiography to determine the distribution of PD-L1 positive tumorsin a mammal or an organ or tissue sample thereof.

In particular, the The ¹⁸F-radiolabeled E01-4PEG-DBCO is useful forpositron emission tomographic (PET) imaging of PD-L1 positive tumors inthe lung, heart, kidneys, liver and skin and other organs of humans andexperimental animals. PET imaging using the ¹⁸F-radiolabeledE01-4PEG-DBCO can be used to obtain the following information:relationship between level of tissue occupancy by candidate PD-L1tumor-treating medicaments and clinical efficacy in patients; doseselection for clinical trials of PD-L1 tumor-treating medicaments priorto initiation of long term clinical studies; comparative potencies ofstructurally novel PD-L1 tumor-treating medicaments; investigating theinfluence of PD-L1 tumor-treating medicaments on in vivo transporteraffinity and density during the treatment of clinical targets with PD-L1tumor-treating medicaments; changes in the density and distribution ofPD-L1 positive tumors during effective and ineffective treatment.

Example 12: Linking of PD-L1 Adnectins to NODAGA to GenerateNODAGA-PD-L1 Adnectins

This Example describes the linking of the E01 and A02 anti-PD-L1Adnectins to NODAGA. As maleimide chemistry is used to link theAdnectins to NODAGA, both Adnectins used a proline followed by acysteine at their C-terminus (as described for E01 above). The aminoacid sequences of the modified E01 and A02 Adnectins are provided in SEQID NOs: 104 and 88, respectively. The cysteine will be used for linkingthe Adnectins to NODAGA. For ⁶⁴Cu labeling of the Adnectins, a 50-foldmolar excess of maleimide-NODAGA (CheMatech) was dissolved in PBS pH 7.4and added to the purified Adnectins in the presence of 1 mM TCEP. FinalDMSO concentrations did not exceed 5% in the conjugation mixtures.Conjugation mixtures were left at room temperature for one hour beforemass spec analysis. After MS confirmation of conjugation, the sampleswere purified by size-exclusion chromatography using a HiLoad 26/60Superdex 75 column (GE Healthcare) equilibrated in PBS pH 7.2.

Example 13: Synthesis of ⁶⁴Cu-Based Anti-PD-L1 Adnectin Probes Synthesisof ⁶⁴Cu-A02-NODAGA

[⁶⁴Cu]-Copper chloride (⁶⁴CuCl₂) in 0.1N hydrochloric acid solution wasneutralized with 0.8 mL of 0.1N sodium acetate (NaOAc) aqueous solutionfor 4 minutes at ambient temperature. 1 mL of the ⁶⁴Cu/NaOAc solutionwas added to A02-NODAGA (30 μL of 1.6 mg/mL) and the crude reaction wasgently pippetted to allow mixing followed by resting at ambienttemperature for 30 minutes. The contents of crude reaction mixture weretransferred to a PD-10 desalting column that was pre-activated with 20mL of 1× phosphate buffered saline (PBS, pH 7.4) buffer prior to loadingof sample. An additional 1.5 mL of 1×PBS was added to the column,followed by an additional 0.8 ml 1×PBS solution and these fractions werediscarded. [⁶⁴Cu]-A02-NODAGA was then collected after a 1.2 mL elutionof the PD-10 column to give 10.79 mCi as the desired product. Qualitycontrol was measured using a reverse phase HPLC system using an AgilentPLRP-S HPLC column Size: 250×4.60 mm, 8 μm, 280 nm and a mobile phase of0.1% Formic Acid in distilled water and acetonitrile. A gradient methodwas used where the percentage of acetonitrile was increased linearlyfrom 10% to 45% over a 30 minute time frame. [⁶⁴Cu]-A02-NODAGA co-elutedwith reference standard at the 22 minute mark of the HPLC chromatogram.Radiochemical purity was measured to be 96% using this method.[⁶⁴Cu]-A02-NODAGA also co-eluted with reference material at the 20minute mark using a size exclusion chromatography, (SEC) Column: GESuperdex 200 GL Size: 10×300 mm, 280 nm. The calculated specificactivity was 956.8 mCi/μmol based on Nanodrop protein concentration andisolated radioactivity of the purified sample.

Procedure for the Synthesis of ⁶⁴Cu-E01-NODAGA

[⁶⁴Cu]-Copper chloride ([⁶⁴Cu]CuCl₂) in 0.1N hydrochloric acid solution(20 mCi in 0.25 mL) was pH adjusted with 1.10 mL of 0.1N ammoniumacetate buffer, then mixed and incubated with E01-NODAGA Adnectin in a1×PBS aqueous solution (40 μL of 1.2 mg/mL, 4.62 nmol) for 30 minutes atambient temperature. After 30 minutes of incubation, the reactionmixture (˜1250 μL) was transferred to the PD-10 desalting column (GEHealthcare Life Science, Sephadex G25 Medium, 14.5×50 mm—equilibratedwith 40 mL of 1×PBS), and the sample was allowed to enter the columncompletely by gravity and followed with 1.1 mL of 1×PBS. After theliquid completely passed through the column, the product was collectedvia elution in 1 mL increments with 1×PBS per sample vial. The⁶⁴Cu-E01-NODAGA was isolated in the second 1 ml fraction and measured tobe 9.26 mCi in 1 ml of 1×PBS. This sample was analyzed using ananalytical size exclusion HPLC method using an Agilent HPLC systemequipped with a UV/vis detector (X=280 nm), a posi-ram detector andSuperdex 200 10/300 GL size-exclusion column (GE Healthcare LifeScience, pore size 13 m). The flow rate was 0.5 mL/min, and the aqueousmobile phase was isocratic with 1×PBS with 0.02% NaN₃ for 60 minutes.The radiochemical purity was 99% using this system, and the productco-eluted with non-radioactive reference standard. Specific activity wascalculated based on the equation of the 3-points calibratio curve(y=656978x). About 100 L of the product solution from vial 3 wasinjected onto the Superdex −200 size exclusion column. Product peak wascollected and measured to be 0.74 mCi, UV counts of product peak was156367 unit, and specific activity was 3.1 mCi/nmol.

Example 14: In Vitro Differentiation of PD-L1-Positive Cells fromPD-L1-Negative Cells with an Anti-PD-L1 Adnectin Imaging Agent

In this experiment, the ⁶⁴Cu-E01 anti-PD-L1 Adnectin (NODAGA was used asa chelator) was tested for its ability to discriminate betweenhPD-L1-positive cells and hPD-L1-negative cells in vitro. Cell labelingwas specific, as evidenced by differential association of ⁶⁴Cu-E01 withhPD-L1-positive L2987 cells compared to hPD-L1-negative HT-29 cells(cell associated radioactivity was 44.6× higher in hPD-L1-positive L2987cells). Specificity was further confirmed as evidenced by a markedreduction in cell-associated ⁶⁴Cu-E01 when co-incubated with excess 450nM cold (unlabeled) E01 Adnectin (99.6% reduction). Cell associated¹⁸F-E01 was minimally reduced (9.9% reduction, not significant) whencells were co-incubated with 450 nM of a cold (unlabeled) non-PD-L1binding Adnectin (FIG. 3).

1×10⁶ hPD-L1-positive L2987 human lung carcinoma cells orhPD-L1-negative HT-29 human colorectal adenocarcinoma cells were placedinto 5 mL culture tubes (n=3 tubes per condition). ⁶⁴Cu-E01 Adnectinsolution was prepared in PBS+0.5% BSA at a concentration of 300 nCi/200μL. Portions of this solution were supplemented with either cold(unlabeled) E01 Adnectin or cold (unlabeled) non-PD-L1 binding Adnectinto a final concentration of 450 nM. Cell samples were centrifuged for 5min at 200×g and then resuspended in 200 μL of the appropriate ⁶⁴Cu-E01Adnectin solution and incubated on ice for 1 hour. After the incubationperiod, cell samples were centrifuged at 200×g and the supernatant wasdiscarded. Cell pellets were resuspended in 1 mL PBS+0.5% BSA and thewash procedure repeated for a total of 3 washes. Following the finalwash, cells were again centrifuged at 200×g and the supernatant wasdiscarded. The radioactivity of the remaining cell pellets was thenmeasured by gamma counter.

Taken together, these results demonstrate the ability of the ⁶⁴Cu-E01Adnectin to differentiate PD-L1(+) vs. PD-L1(−) cells in vitro.Specificity was further demonstrated by a marked reduction incell-associated radiotracer in samples co-incubated with 450 nMunlabeled anti-PD-L1 E01 Adnectin (and only a statisticallyinsignificant reduction when co-incubated with 450 nM of a non-PD-L1binding Adnectin). Similar experiments using different Adnectin variantsas well as ¹⁸F as the radionuclide were conducted, with similar results.

Example 15: Distinguishing PD-L1-Positive Tumors from PD-L1-NegativeTumors with an Anti-PD-L1 Adnectin Imaging Agent

For PET imaging, rapid blood clearance rates provide an advantage overmore slowly clearing proteins, such as antibodies, by minimizing theamount of time needed for “background” probe signals to deplete fromnon-relevant tissue. In the clinic, long blood half-lifeantibody-based-PET tracers may require several days of waiting postinjection before images can be collected. Rapid clearing probes open thedoor to high contrast images that can be collected on the same day theprobe is injected, and very importantly, they can also serve to reduceoverall radiation exposure to the animals studied or patients examined.

In this experiment, the ⁶⁴Cu-A01 anti-PD-L1 Adnectin (NODAGA was used asthe chelator), produced as described in the above Examples, was testedfor its ability to discriminate between hPD-L1-positive tumors andhPD-L1-negative tumors in mice.

Mice bearing bilateral xenograft tumors were produced by introducing1×10⁶ hPD-L1(+) L2987 human lung carcinoma cells and 1.5×10⁶ hPD-L1(−)HT-29 human colon carcinoma cells subcutaneously on opposite sides ofthe mouse. Once tumors reached approximately 300 mm³ (approximately 2-3weeks after cell implantation), animals were selected for imaging. Forimaging, animals were placed under anesthesia with 2% isoflurane andtail vein catheters were installed. Mice were then placed into a customanimal holder with capacity for 4 animals, where they remained underanesthesia for the duration of the study. The animal holder wastransferred to the microPET® F120™ scanner (Siemens PreclinicalSolutions, Knoxville, Tenn.). The axial field of view of this instrumentis 7.6 cm. With this limitation, animals were positioned such that thescanning region was from immediately in front of the eyes toapproximately the base of the tail.

A 10-minute transmission image was first acquired using a ⁵⁷Co pointsource for the purpose of attenuation correction of the final PETimages. Following the transmission scan, radiotracer solutions wereadministered via the previously installed tail vein catheters and a 2hour emission image was acquired. Injected radiotracer solutionsconsisted of either approximately 200 μCi ⁶⁴Cu-A02 (with a NODAGAchelator) or 200 μCi ⁶⁴Cu-A02 supplemented with 3 mg/kg finalconcentration of cold, unlabeled A02 Adnectin (based on individualanimal weight). All injections were formulated in 200 L saline prior toinjection. Exact injected doses were calculated by taking directmeasurements of the formulated dose and subtracting the radioactivityremaining in the syringe and the tail vein catheter.

Images were reconstructed using a maximum a posteriori (MAP) algorithmwith attenuation correction using the collected transmission images andcorrected for radioisotope decay. In the final images, regions ofinterest (ROIs) were drawn around the tumor boundary using ASIProsoftware (Siemens Preclinical Solutions). Time-activity curves werecalculated for each ROI to yield a quantitative view of radiotracerwithin the tumor volume over the course of the 2 hour emission image.For final comparison, individual time-activity curves were normalizedbased on the injected radiotracer dose for each specific animal.Radiotracer uptake was compared across tumors using the final 10 minutesof each time-activity curve (1 hour 50 minutes—2 h post-radiotracerinjection). Using this methodology, radiotracer uptake in hPD-L1(+)L2987 xenografts was 3.05× that seen hPD-L1(−) HT-29 xenografts inanimals receiving only the ⁶⁴Cu-A02 radiotracer. In animals co-injectedwith the ⁶⁴Cu-A02 radiotracer and 3 mg/kg unlabeled A02 Adnectin uptakein the hPD-L1(+) L2987 xenografts was only 1.04× that seen in hPD-L1(−)HT-29 xenografts (FIGS. 4A and 4B).

For some studies, animals were sacrificed via cervical dislocationimmediately following imaging. Necropsy was then performed on theanimals, and individual tissues were collected (blood, heart, lung,liver, spleen, kidney, muscle, stomach, bone, L2987 tumor, and HT-29tumor) into pre-weighed tubes. All tissues were then weighed again todetermine the weight of each tissue. The radioactivity in each tissuewas then directly measured ex vivo using a Perkin-Elmer Wizard3 gammacounter. For all tissues, measured values in counts per minute (CPM)were normalized to the injected radioactive dose for the individualanimals and corrected for radioactive decay. These results were thenplotted to show the biodistribution of the radiotracer. An example ofthis analysis for the ¹⁸F-A02 Adnectin radiotracer is shown in FIG. 5.These results demonstrate clear differential uptake of the radiotracerin hPD-L1(+) L2987 xenografts compared to hPD-L1(−) HT-29 xenografts.Furthermore, the only tissue with higher PD-L1 uptake was the kidney,which is expected as clearance of the ¹⁸F-A02 Adnectin is expected to bevia kidney filtration based on the molecular weight of the molecule.

Taken together, these results provide direct visualization ofdifferentiation of hPD-L1(+) versus hPD-L1(−) xenograft tumors in vivo.Specificity was further demonstrated by co-injection of 3 mg/kgunlabeled anti-PD-L1 A02 Adnectin, resulting in a reduction ofradiotracer uptake in hPD-L1(+) tumors to the level of hPD-L1(−)xenografts. This further validates the use of anti-PD-L1 Adnectins forvisualization of PD-L1 tissue expression using PET imaging. Similarexperiments using ¹⁸F as the radionuclide were conducted in mice, andsimilar results were obtained, reaching a maximum radiotracer uptakeratio of 3.53:1 in hPD-L1(+) L2987 xenografts vs. hPD-L1(−) HT-29xenografts using the ¹⁸F-A02 Adnectin radiotracer.

The anti-PD-L1 Adnecin-based imaging agents also showed similar resultswhen performed in cynomolgus monkeys. In these studies, the ¹⁸F-E01anti-PD-L1 Adnectin, produced as described in the above Examples, wastested for its ability to produce high-contrast images in cynomolgusmonkeys. The anti-PD-L1 Adnectins described here maintain high affinityfor cynomolgus PD-L1 (but have low affinity for rodent PD-L1).Furthermore, as cynomolgus monkeys do not contain PD-L1(+) tumors as inmouse models, imaging performance was assessed primarily on thebackground levels measured in the images in the context of endogenousPD-L1 expression (with low background enabling the potential forhigh-sensitivity detection of PD-L1(+) tissues). In these studies,background levels in the resulting PET images were very low, withnotable radiotracer accumulation noted mainly in the kidneys, spleen,and bladder.

Cynomolgus male monkeys with a previously installed vascular access port(VAP) were anesthetized with 0.02 mg/kg atropine, 5 mg/kg Telazol and0.01 mg/kg buprenorphine I.M. (all drawn into a single syringe). An i.v.catheter is then placed in the cephalic vessel for fluid administrationduring the imaging procedure to maintain hydration. Animals wereintubated with an endotracheal tube—usually 3.0 mm and transferred tothe imaging bed of a microPET® F220™ PET instrument (Siemens PreclinicalSolutions, Knoxville, Tenn.). Anesthesia was maintained with isofluraneand oxygen and I.V. fluids (LRS) were administered at a rate of 6ml/kg/hr during the imaging procedure. As the axial field of view of themicroPET® F220™ instrument is only 7.6 cm, images over 5 distinct bedpositions were acquired to create a composite image of the animals fromjust above the heart through approximately the pelvis.

For each field of view, a 10 minute transmission image was firstacquired using a ⁵⁷Co point source for the purpose of attenuationcorrection of the final PET images. Once transmission images wereacquired for all bed positions, approximately 1.5 mCi (approximately0.015 mg/kg) of the ¹⁸F-E01 Adnectin radiotracer was administered viathe installed VAP. 5 minute duration emission scans were thensequentially acquired for each bed position, beginning at position 1centered approximately at the heart and moving toward the pelvis of theanimal. Once images were acquired at each position (1 through 5), theimaging bed was moved back to bed position 1 and the process wasrepeated. Using this procedure, a total of 5 distinct images wereacquired for each bed position over the duration of the imaging study.

Individual images were reconstructed using a filtered back projection(FBP) algorithm with attenuation correction using the collectedtransmission images and corrected for radioisotope decay. Finalcomposite images were then produced by aligning images from all 5 bedpositions obtained from a single pass (i.e. a single composite image wasproduced from each set of sequential images from bed positions 1 through5) covering the duration of the imaging study. Final images werevisually inspected to note areas of visible radiotracer uptake (i.e.spleen, kidney, bladder) and background tissue (muscle) (FIG. 6).Background accumulation of ¹⁸F-E01 Adnectin was very low, with littlesignal visible in background tissues such as muscle. Additionally,uptake was verified in the spleen, which is believed to be PD-L1(+)based on mRNA expression. Thus, studies in cynomolgus monkeysdemonstrate the potential for high-sensitivity PD-L1 imaging in thecontext of endogenous PD-L1.

In aggregate, PET studies in rodent and cynomolgus monkey show that ⁶⁴Cuand ¹⁸F labeled anti-human PD-L1 Adnectins provide strong and specificprobes for in vivo labeling of PD-L1 positive tissues with the potentialfor high-sensitivity detection of tissues with low level PD-L1expression.

In vivo imaging experiments were also conducted with an anti-PD-L1antibody, and the areas that this imaging agent detected were the sameareas that were detected with the PD-L1 imaging agent, thereforeconfirming that anti-PD-L1 Adnectin imaging agents successfully detectPD-L1 positive cells in vivo.

Example 16: In Vitro Autoradiography of Human and Xenograft Tissue with[¹⁸F]-E01 Anti-PD-L1 Adnectin

Human lung tumor tissues were embedded in OCT and chilled in2-methylbutane for 2-5 minutes until frozen. Samples were stored in −80°C. degree freezer until use. Human xenograft tissues were also includedin the assay. Mice bearing bilateral xenografts were produced byintroducing 4×10⁶ hPD-L1(+) L2987 cells and 1.5×10⁶ hPD-L1(−) HT-29 tcells subcutaneously into opposite flanks of nu/nu mice. Once resultingxenograft tumors reached appropriate size (approx. 200-300 mm³), micewere anesthetized with 2% isoflurane and sacrificed via cervicaldislocation. Fresh tumor tissues were excised, immersed into OCT andchilled in 2-methylbutane for 2-5 minutes until frozen. The tissues werethen wrapped in foil/ZIPLOC® bag and stored at −80° C. until use. Forall tissues (human lung tumor and xenografts) sections of 5 μm thickness(collected as 2 sections/slide) were cut using a cryostat, thaw-mountedon glass microscope slides, and allowed to air dry for approximately 30minutes.

Blocking studies with cold (unlabeled) A02 Adnectin at 0.025 nM, 0.25nM, 2.5 nM and 25 nM respectively and 25 nM non-PD-L1 binding Adnectinwere conducted using the following conditions. The individual slides, 1slide per concentration, were placed in plastic slide cassettes andpre-incubated in Dako serum-free protein block solution for 30 minutes.Slides were then transferred to glass slide incubation chambers forfurther incubation. Separately, a stock solution of 0.25 nM ¹⁸F-A02Adnectin was produced by diluting 10.6 μl of the original stockradioligand solution (7064 nM at the time of experiment) with 300 ml ofPBS+0.5% BSA. From this stock solution, 40 ml was added to eachincubation chamber. One of these chambers contained only the radioligandbuffer solution, which is referred to as the total binding section.Other incubation chambers received 40 ml of this stock solution alongwith the relevant concentration of blocking compound (unlabeled A02Adnectin at 0.025 nM, 0.25 nM, 2.5 nM, or 25 nM or unlabeled non-PD-L1binding Adnectin at 25 nM). Slides were incubated in the individualbuffer solutions for 1 hour at room temperature to reach maximumbinding. After incubation, slides from each treatment group were removedfrom the incubation solutions and placed in an ice-cold wash buffer(PBS+0.5% BSA) for 3 minutes and rinsed 4 separate times. Slides werethen dried under a stream of cold air for approximately 30 minutes. Theair-dried slides were exposed by placing the slides onto an imagingplate (BAS-SR 3545S) overnight at room temperature. The imaging platewas scanned using the bioimaging analyzer (Fujifilm Fluorescent ImageAnalyzer, FLA-9000). The pixel size of the autoradiogram images was 100μm. Image analysis was performed using the Multi-Gauge software. Theregions of interest (ROIs) were drawn to surround the entire tumortissue in all study groups. Autogradiography signal fromtissue-associated radioactivity was quantified from these ROIs.

The apparent displacement of the ¹⁸F-A02 Adnectin radioligand whencompared to the total binding sections was determined for 4 differentconcentrations (0.025 nM, 0.25 nM, 2.5 nM and 25 nM) of unlabeled A02Adnectin in both human lung tumor sections as well as human xenograftsections. A dose dependent displacement of ¹⁸F-A02 was seen in alltissue sections with the addition of unlabeled A02 Adnectin, while 25 nMnon-PD-L1 binding Adnectin showed minimal blockade in all tissuescompared to total binding (FIG. 7).

Serial 5 μm tissue sections from each tissue were subjected to ananti-human-PD-L1 immunohistochemical procedure to verify the level ofPD-L1 antigen expression in the samples (FIG. 8).

Taken together, these results provide direct visualization of PD-L1 inboth human lung tumor samples as well as human xenograft tissues. Thelevel of radioligand binding in the individual tissues corresponds withthe intensity of PD-L1 staining of frozen sections by IHC. In addition,the dose dependent blockade of the receptor with unlabeled anti-PD-L1A02 Adnectin (and lack of blockade with unlabeled non-PD-L1 bindingAdnectin), further validates the use of ¹⁸F-A02 for visualization ofPD-L1 tissue expression using PET imaging.

Example 17: Automated preparation of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridineaccording to the general procedure for radiosynthesis using commercialGE TRACERlab FX2 N synthesis unit

Procedure:

The automated synthesis of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridinewas carried out using a non-cassette type GE TRACERlab FX2 N Synthesismodule. The setup of the synthesis unit is summarized in Table 4. Theaqueous [¹⁸F]-Fluoride solution (2.0 ml, 29.6 GBq/800 mCi) was deliveredto a Sep-Pak light QMA [The Sep-Pak light QMA cartridge waspre-conditioned sequentially with 5 ml of 0.5 M potassium bicarbonate, 5ml of deionized water, and 5 ml of acetonitrile before use.] Uponcompletion of this transfer, the aqueous [¹⁸F] fluoride was releasedfrom the QMA Sep-Pak by the addition of the elution mixture (from “V”)into the reactor. The solvent was evaporated under a gentle stream ofnitrogen and vacuum. The solution of precursor (from “V3”) was added tothe dried cryptand residue and this reaction mixture was heated 120° C.for 10 minutes. Then 4 ml of distilled water (from “V4”) was added tothe crude reaction mixture in the reactor and the mixture wastransferred to the 5 ml sample injection loop of the semi-preparativeHPLC via a liquid sensor which controls the end of the loading. Themixture was loaded onto the semi-preparative HPLC column (Luna C18(2).250×10 mm, Phenomenex). A mixture of 35% acetonitrile in an aqueous 0.1%trifluoroacetic acid solution was flushed through the column at a rateof 4.6 ml per minute. The product was collected from this HPLC columninto the dilution flask which contained 15 ml distilled water and itsentire contents were transferred to a tC18 1 gram, solid phaseextraction cartridge. 352 mCi (13 GBq) of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridinewas released from this cartridge (from “V14”) with 3 ml of ethanol andmay be used to generate ¹⁸F labeled biologic products by takingadvantage of “click” azide-alkyne reaction with the appropriate biologiccontaining an alkynes.

TABLE 4 Vial 1 (V1) 16 mg K.2.2.2, 3 mg Potassium carbonate, dissolvedin 0.1 ml of distilled water and 1.4 ml of acetonitrile Vial 3 (V3) 2 mg3-(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine in 0.5ml DMSO Vial 4 (V4) 4 ml of distilled water Vial 14 (V14) 3 ml of 100%ethanol Dilution Flask 15 ml of distilled water Cartridge 1 (C1) tC18 6cc 1 g sep pack HPLC Column Luna C18(2), 250 × 10 mm, 5 □m, PhenomenexHPLC Solvent 35% acetonitrile in an aqueous 0.1% trifluoroacetitic acidsolution HPLC flow 4.6 ml/min

Example 18 Automated preparation of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridineaccording to the general procedure for radiosynthesis on IBA Syntherasynthesis unit

Procedure:

The automated synthesis of[F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine wascarried out using a cassette type IBA Synthera Synthesis module and anappropriately assembled integrator fluidic processor kit. The integratorfluidic processor (IFP) kit was loaded with appropriate precursors forthis synthesis and is summarized in Table 5. The purification wasperformed on an Varian HPLC unit. The filling of the injection loop ofthe HPLC was controlled by a steady stream of nitrogen on the HPLC unit.The setup of both automates are summarized in Table 2. The aqueous[¹⁸F]-Fluoride solution (2.0 ml, 29.6 GBq/800 mCi) was delivered to aSep-Pak light QMA [The Sep-Pak light QMA cartridge was pre-conditionedsequentially with 5 ml of 0.5 M potassium bicarbonate, 5 ml of deionizedwater, and 5 ml of acetonitrile before use.] Upon completion of thistransfer, the aqueous [¹⁸F] fluoride was released from the QMA Sep-Pakby the addition of the elution mixture (from “V”) into the reactor. Thesolvent was evaporated under a gentle stream of nitrogen and vacuum. Thesolution of precursor (from “V2”) was added to the dried cryptandresidue and this reaction mixture was heated 120° C. for 10 minutes.Then 3 ml of distilled water (from “V4”) was added to the crude reactionmixture in the reactor and the mixture is transferred to the 5 ml sampleinjection loop of the semi-preparative HPLC via a liquid sensor whichcontrols the end of the loading. The mixture was loaded onto thesemi-preparative HPLC column (Luna C18(2). 250×10 mm, Phenomenex). Amixture of 35% acetonitrile in an aqueous 0.1% trifluoroacetic acidsolution was flushed through the column at a rate of 4.6 ml per minute.The product was collected from this HPLC column into the dilution flaskwhich contained 15 ml distilled water and its entire contents weretransferred to a tC18 1 gram, solid phase extraction cartridge. 325 mCi(12 GBq) of[¹⁸F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridinewas released from this cartridge with 3 ml of ethanol and may be used togenerate ¹⁸F labeled biologic products by taking advantage of “click”azide-alkyne reaction with the appropriate biologic containing analkynes.

TABLE 5 Vial 1 (V1) 22 mg K.2.2.2, 4 mg Potassium carbonate, dissolvedin 0.3 ml of distilled water and 0.3 ml of acetonitrile Vial 2 (V2) 2 mg3-(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine in 0.5ml DMSO Vial 4 (V4) 3 ml of distilled water Dilution Flask 15 ml ofdistilled water Cartridge 1 (C1) tC18 6 cc 1 g sep pack HPLC Column LunaC18(2), 250 × 10 mm, 5 □m, Phenomenex HPLC Solvent 35% acetonitrile inan aqueous 0.1% trifluoroacetitic acid solution HPLC flow 4.6 ml/min

Example 19: Synthesis of ⁶⁸Ga-Based Anti-PD-L1 Adnectin Probes Synthesisof ⁶⁸Ga-E01-NODAGA

[⁶⁸Ga]-Gallium chloride in 0.1N hydrochloric acid solution wasneutralized with 32 mg of sodium acetate (NaOAc) for 4 minutes atambient temperature, the resultant solution was stirred to ensure theentire volume was properly mixed. This solution was then added toE01-NODAGA (15 μL of 1.3 mg/mL) solution and the crude reaction wasgently pipetted to allow mixing followed by resting at ambienttemperature for 15 minutes. The contents of crude reaction mixture weretransferred to a PD-10 desalting column that was pre-activated with 20mL of 1× phosphate buffered saline (PBS, pH 7.4) buffer prior to loadingof sample. An additional 1.5 mL of 1×PBS was added to the column,followed by an additional 0.8 ml 1×PBS solution and these fractions werediscarded. [⁶⁸Ga]-E01-NODAGA was then collected after a 1.4 mL elutionof the PD-10 column to give 5.78 mCi (214 MBq) as the desired product.Quality control was measured using a reverse phase HPLC system using anAgilent PLRP-S HPLC column Size: 250×4.60 mm, 8 μm, 280 nm and a mobilephase of 0.1% Formic Acid in distilled water and acetonitrile. Agradient method was used where the percentage of acetonitrile wasincreased linearly from 10% to 45% over a 30 minute time frame.[⁶⁸Ga]-E01-NODAGA co-eluted with reference standard at the 22 minutemark of the HPLC chromatogram. Radiochemical purity was measured to be98% using this method. [⁶⁸Ga]-E01-NODAGA also co-eluted with referencematerial at the 20 minute mark using a size exclusion chromatography,(SEC) Column: GE Superdex 200 GL Size: 10×300 mm, 280 nm.

 1 MGVSD VPRDL EVVAA TPTSL LISWR AQLSP SFYYR ITYGE 41  TGGNS PVQEF TVPND VMTAT ISGLK PGVDY TITVY AVTTH 81GVYFY SPISI NYRTP CHHHH HH 

TABLE 3 SEQUENCE LISTING SEQ ID DESCRIPTION SEQUENCE 1Full length wild-type VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNShuman ¹⁰Fn3 domain PVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSK PISINYRT2 Core wild-type human EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVP¹⁰Fn3 domain GSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT 3Core ¹⁰Fn3-based scaffoldEVVAA(Z)_(a)LLISW(Z)_(x)YRITY(Z)_(b)FTV(Z)_(y)ATISGL(Z)_(c)Ywith variable AB, BC, CD, TITVYA(Z)_(z)ISINYRT DE, EF, and FG loops 4Core ¹⁰Fn3-based scaffoldEVVAATPTSLLISW(Z)_(x)YRITYGETGGNSPVQEFTV(Z)_(y)ATISGwith variable BC, DE, and LKPGVDYTITVYA(Z)_(z)ISINYRT FG loops 5ATI-968 core (aka EVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVQEFTVPGTGADX_1760_C01) YTATISGLKPGVDYTITVYAVTDGASIASYAFPISINYRT 6 ATI-968 BC loopIAPFYNVIY 7 ATI-968 DE loop PGTGYT 8 ATI-968 FG loop VTDGASIASYAFP 9ATI-968 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVQEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISIN YRT 10ATI-968 w/N leader + his GVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVtag QEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISIN YRTHHHHHH 11ATI-968 w/N leader and C GVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVtail QEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISIN YRTEIDKPSQ 12ATI-968 w/N leader and C GVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVtail + his tag QEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISINYRTEIDKPSQHHHHHH 13 ATI-968 w/N leader andGVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPV modified C-terminusQEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISIN including PC YRTPC 14ATI-968 w/N leader and GVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVmodified C-terminus QEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISINincluding PC + his tag YRTPCHHHHHH 15 ATI-968-full lengthMGVSDVPRDLEVVAATPTSLLISWIAPFYNVIYYRITYGETGGNSPVQEFTVPGTGYTATISGLKPGVDYTITVYAVTDGASIASYAFPISI NYRTEIDKPSQHHHHHH 16ATI-968-core (nucleotide GAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGATCGsequence) CTCCGTTCTACAATGTCATCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTACTGGTTATACAGCTACAATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGATGGAGCATCCATTGCTTCATACGCGTTTCCAATTTCCATTAATTACCGCACA 17 ATI-968 w/N leaderATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA (nucleotide sequence withCCCCCACCAGCCTGCTGATCAGCTGGATCGCTCCGTTCTACAATGT N-terminal methionine)CATCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTACTGGTTATACAGCTACAATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGATGGAGCATCCATTGCTTCATACGCGTTTCCAATTTCCATT AATTACCGCACA 18ATI-968 w/N leader and ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAmodified C-terminus CCCCCACCAGCCTGCTGATCAGCTGGATCGCTCCGTTCTACAATGTincluding PC (nucleotide CATCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTsequence with N-terminal GTCCAGGAGTTCACTGTGCCTGGTACTGGTTATACAGCTACAATCAmethionine) GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGATGGAGCATCCATTGCTTCATACGCGTTTCCAATTTCCATT AATTACCGCACACCGTGC 19ATI-968 w/N leader and C ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAtail + his tag (nucleotideCCCCCACCAGCCTGCTGATCAGCTGGATCGCTCCGTTCTACAATGT sequence with N-terminalCATCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT methionine)GTCCAGGAGTTCACTGTGCCTGGTACTGGTTATACAGCTACAATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGATGGAGCATCCATTGCTTCATACGCGTTTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACC ACCACTGA 20ATI-964 core (parent of EVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVQEFTVPPDQADX_5322_A02) KTATISGLKPGVDYTITVYAVRLEEAHYYRESPISINYRT 21ATI-964 BC loop SYDGSIERY 22 ATI-964 DE loop PPDQKT 23 ATI-964 FG loopVRLEEAHYYRESP 24 ATI-964 w/N leaderGVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISIN YRT 25ATI-964 w/N leader + his GVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVtag QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISIN YRTHHHHHH 26ATI-964 w/N leader and C GVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVtail QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISIN YRTEIDKPSQ 27ATI-964 w/N leader and C GVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVtail + his tag QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISINYRTEIDKPSQHHHHHH 28 ATI-964 w/N leader andGVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPV modified C-terminusQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISIN including PC YRTPC 29ATI-964 w/N leader and GVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVmodified C-terminus QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISINincluding PC + his tag YRTPCHHHHHH 30 ATI-964-full lengthMGVSDVPRDLEVVAATPTSLLISWSYDGSIERYYRITYGETGGNSPVQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYYRESPISI NYRTEIDKPSQHHHHHH 31ATI-964-core (nucleotide GAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTTsequence) ACGACGGTTCGATTGAACGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCAGGCTGGAAGAAGCTCATTACTATCGAGAGTCTCCAATTTCCATTAATTACCGCACA 32 ATI-964 w/N leaderATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA (nucleotide sequence withCCCCCACCAGCCTGCTGATCAGCTGGTCTTACGACGGTTCGATTGA N-terminal methionine)ACGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCAGGCTGGAAGAAGCTCATTACTATCGAGAGTCTCCAATTTCCATT AATTACCGCACA 33ATI-964 w/N leader and ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAmodified C-terminus CCCCCACCAGCCTGCTGATCAGCTGGTCTTACGACGGTTCGATTGAincluding PC (nucleotide ACGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTsequence with N-terminal GTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAmethionine) GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCAGGCTGGAAGAAGCTCATTACTATCGAGAGTCTCCAATTTCCATT AATTACCGCACACCGTGC 34ATI-964 w/N leader and C ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAtail + his tag (nucleotideCCCCCACCAGCCTGCTGATCAGCTGGTCTTACGACGGTTCGATTGA sequence with N-terminalACGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT methionine)GTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCAGGCTGGAAGAAGCTCATTACTATCGAGAGTCTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACC ACCACTGA 35 ATI-965 coreEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVQEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISINYRT 36 ATI-965 BC loop TAYDSVDKY 37ATI-965 DE loop GPRHHT 38 ATI-965 FG loop VYHTEPGYHAHMP 39ATI-965 w/N leader GVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVQEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISIN YRT 40ATI-965 w/N leader + his GVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVtag QEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISIN YRTHHHHHH 41ATI-965 w/N leader and C GVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVtail QEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISIN YRTEIDKPSQ 42ATI-965 w/N leader and C GVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVtail + his tag QEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISINYRTEIDKPSQHHHHHH 43 ATI-965 w/N leader andGVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPV modified C-terminusQEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISIN including PC YRTPC 44ATI-965 w/N leader and GVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVmodified C-terminus QEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHAHMPISINincluding PC + his tag YRTPCHHHHHH 45 ATI-965-full lengthMGVSDVPRDLEVVAATPTSLLISWTAYDSVDKYYRITYGETGGNSPVQEFTVGPRHHTATISGLKPGVDYTITVYAVYHTEPGYHA HMPISINYRTEIDKPSQHHHHHH46 ATI-965-core (nucleotideGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGACTG sequence)CATACGACTCTGTTGACAAATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGGCCCTAGACATCACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCTATCACACTGAACCGGGCTATCATGCTCATATGCCAATTTCCATTAATTACCGCACA 47 ATI-965 w/N leaderATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA (nucleotide sequence withCCCCCACCAGCCTGCTGATCAGCTGGACTGCATACGACTCTGTTGA N-terminal methionine)CAAATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGGCCCTAGACATCACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCTATCACACTGAACCGGGCTATCATGCTCATATGCCAATTTCCATT AATTACCGCACA 48ATI-965 w/N leader and ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAmodified C-terminus CCCCCACCAGCCTGCTGATCAGCTGGACTGCATACGACTCTGTTGAincluding PC (nucleotide CAAATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTsequence with N-terminal GTCCAGGAGTTCACTGTGGGCCCTAGACATCACACAGCTACCATCAmethionine) GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCTATCACACTGAACCGGGCTATCATGCTCATATGCCAATTTCCATT AATTACCGCACACCGTGC 49ATI-965 w/N leader and C ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAtail + his tag (nucleotideCCCCCACCAGCCTGCTGATCAGCTGGACTGCATACGACTCTGTTGA sequence with N-terminalCAAATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT methionine)GTCCAGGAGTTCACTGTGGGCCCTAGACATCACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCTATCACACTGAACCGGGCTATCATGCTCATATGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACC ACCACTGA 50 ATI-966 coreEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPVQEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRT 51 ATI-966 BC loop HRFSSIMAY 52ATI-966 DE loop AGSVNT 53 ATI-966 FG loop VTIHNVSFP 54ATI-966 w/N leader GVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPVQEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRT 55 ATI-966 w/N leader +his GVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPV tagQEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRTH HHHHH 56ATI-966 w/N leader and C GVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPVtail QEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRTE IDKPSQ 57ATI-966 w/N leader and C GVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPVtail + his tag QEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRTEIDKPSQHHHHHH 58 ATI-966 w/N leader andGVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPV modified C-terminusQEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRTP including PC C 59ATI-966 w/N leader and GVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPVmodified C-terminus QEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRTPincluding PC + his tag CHHHHHH 60 ATI-966-full lengthMGVSDVPRDLEVVAATPTSLLISWHRFSSIMAYYRITYGETGGNSPVQEFTVAGSVNTATISGLKPGVDYTITVYAVTIHNVSFPISINYRT EIDKPSQHHHHHH 61ATI-966-core (nucleotide GAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGCATAsequence) GGTTCTCTTCTATCATGGCGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGCTGGCTCTGTTAACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACGATCCATAACGTTTCTTTCCCAATTTC CATTAATTACCGCACA 62ATI-966 w/N leader ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA(nucleotide sequence with CCCCCACCAGCCTGCTGATCAGCTGGCATAGGTTCTCTTCTATCATN-terminal methionine) GGCGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGCTGGCTCTGTTAACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACGATCCATAACGTTTCTTTCCCAATTTCCATTAATTACCGCACA 63 ATI-966 w/N leader andATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA modified C-terminusCCCCCACCAGCCTGCTGATCAGCTGGCATAGGTTCTCTTCTATCAT including PC (nucleotideGGCGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT sequence with N-terminalGTCCAGGAGTTCACTGTGGCTGGCTCTGTTAACACAGCTACCATCA methionine)GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACGATCCATAACGTTTCTTTCCCAATTTCCATTAATTACCGCACA CCGTGC 64ATI-966 w/N leader and C ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAtail + his tag (nucleotideCCCCCACCAGCCTGCTGATCAGCTGGCATAGGTTCTCTTCTATCAT sequence with N-terminalGGCGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT methionine)GTCCAGGAGTTCACTGTGGCTGGCTCTGTTAACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACGATCCATAACGTTTCTTTCCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCACTGA 65 ATI-967 core (parent ofEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPVQEFTVPVAS ADX_5417_E01)GTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYRT 66 ATI-967 BC loop QGQLSPSFY 67ATI-967 DE loop PVASGT 68 ATI-967 FG loop VTSHGIYFYAP 69ATI-967 w/N leader GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPVQEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYR T 70 ATI-967 w/N leader +his GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPV tagQEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYR THHHHHH 71ATI-967 w/N leader and C GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPVtail QEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYR TEIDKPSQ 72ATI-967 w/N leader and C GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPVtail + his tag QEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYRTEIDKPSQHHHHHH 73 ATI-967 w/N leader andGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPV modified C-terminusQEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYR including PC TPC 74ATI-967 w/N leader and GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPVmodified C-terminus QEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINYRincluding PC + his tag TPCHHHHHH 75 ATI-967-full lengthMGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYYRITYGETGGNSPVQEFTVPVASGTATISGLKPGVDYTITVYAVTSHGIYFYAPISINY RTEIDKPSQHHHHHH 76ATI-967-core (nucleotide GAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGCAGGsequence) GACAGCTGTCTCCGTCTTTCTATTACCGAATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGCTAGTGGGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTCTCATGGCATATACTTCTACGCTCC AATTTCCATTAATTACCGCACA 77ATI-967 w/N leader ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA(nucleotide sequence with CCCCCACCAGCCTGCTGATCAGCTGGCAGGGACAGCTGTCTCCGTCN-terminal methionine) TTTCTATTACCGAATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGCTAGTGGGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTCTCATGGCATATACTTCTACGCTCCAATTTCCATTAATTAC CGCACA 78ATI-967 w/N leader and ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAmodified C-terminus CCCCCACCAGCCTGCTGATCAGCTGGCAGGGACAGCTGTCTCCGTCincluding PC (nucleotide TTTCTATTACCGAATCACTTACGGCGAAACAGGAGGCAATAGCCCTsequence with N-terminal GTCCAGGAGTTCACTGTGCCTGTTGCTAGTGGGACAGCTACCATCAmethionine) GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTCTCATGGCATATACTTCTACGCTCCAATTTCCATTAATTAC CGCACACCGTGC 79ATI-967 w/N leader and C ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAtail + his tag (nucleotideCCCCCACCAGCCTGCTGATCAGCTGGCAGGGACAGCTGTCTCCGTC sequence with N-terminalTTTCTATTACCGAATCACTTACGGCGAAACAGGAGGCAATAGCCCT methionine)GTCCAGGAGTTCACTGTGCCTGTTGCTAGTGGGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTCTCATGGCATATACTTCTACGCTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCACT GAT 80 ADX_5322_A02 coreEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPVQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISINYRT 81 ADX_5322_A02 BC loopSYDGPIDRY 82 ADX_5322_A02 DE loop PPDQKT 83 ADX_5322_A02 FG loopVRLEEAHYNREFP 84 ADX_5322_A02 w/NGVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPV leaderQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISIN YRT 85 ADX_5322_A02 w/NGVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPV leader + his tagQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISIN YRTHHHHHH 86ADX_5322_A02 w/N GVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPVleader and C tail QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISINYRTEIDKPSQ 87 ADX_5322_A02 w/NGVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPV leader and C tail +his tag QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISIN YRTEIDKPSQHHHHHH88 ADX_5322_A02 w/N GVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPVleader and modified C- QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISINterminus including PC YRTPC 89 ADX_5322_A02 w/NGVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPV leader and modified C-QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISIN terminus including PC +YRTPCHHHHHH his tag 90 ADX_5322_A02-Mal-GVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPV DBCO-FFPF18QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISINYRTPC-[Maleamide-DBCO-FFP(18F)] 91 ADX_5322_A02 fullMGVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSP lengthVQEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISI NYRTPCHHHHHH 92ADX_5322_A02-core GAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTT(nucleotide sequence) ACGATGGCCCAATTGACCGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCCGGCTGGAAGAAGCTCATTACAATCGAGAGTTTCCAATTTCCATTAATTACCGCACA 93 ADX_5322_A02 w/NATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCA leader (nucleotideCCCCCACCAGCCTGCTGATCAGCTGGTCTTACGATGGCCCAATTGA sequence with N-terminalCCGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT methionine)GTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCCGGCTGGAAGAAGCTCATTACAATCGAGAGTTTCCAATTTCCATT AATTACCGCACA 94ADX_5322_A02 w/N ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAleader and modified C- CCCCCACCAGCCTGCTGATCAGCTGGTCTTACGATGGCCCAATTGAterminus including PC CCGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT(nucleotide sequence with GTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAN-terminal methionine) GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCCGGCTGGAAGAAGCTCATTACAATCGAGAGTTTCCAATTTCCATT AATTACCGCACACCGTGC 95ADX_5322_A02 w/N ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAleader and C tail + his tagCCCCCACCAGCCTGCTGATCAGCTGGTCTTACGATGGCCCAATTGA (nucleotide sequence withCCGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT N-terminal methionine)GTCCAGGAGTTCACTGTGCCTCCGGATCAGAAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCCGGCTGGAAGAAGCTCATTACAATCGAGAGTTTCCAATTTCCATTAATTACCGCACACCGTGCCACCATCACCACCACCACTGA 96 ADX_5417_E01 coreEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPVQEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYRT 97 ADX_5417_E01 BC loop RAQLSPSFY98 ADX_5417_E01 DE loop PNDVMT 99 ADX_5417_E01 FG loop VTTHGVYFYSP 100ADX_5417_E01 w/N GVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPV leaderQEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYR T 101 ADX_5417_E01 w/NGVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPV leader + his tagQEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYR THHHHHH 102ADX_5417_E01 w/N GVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPVleader and C tail QEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYRTEIDKPSQ 103 ADX_5417_E01 w/NGVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPV leader and C tail + hisQEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYR tag TEIDKPSQHHHHHH 104ADX_5417_E01 w/N GVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPVleader and modified C- QEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYRterminus including PC TPC 105 ADX_5417_E01 w/NGVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPV leader and modified C-QEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYR terminus including PC +TPCHHHHHH his tag 106 ADX-5417_E01-Mal-GVSDVPRDLEVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPV DBCO-FFPF18QEFTVPPDQKTATISGLKPGVDYTITVYAVRLEEAHYNREFPISINYRTPC-[Maleamide-DBCO-FFP(18F)] 107 ADX-5417_E01 full lengthMGVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPVQEFTVPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINY RTPCHHHHHH 108ADX-5417_E01-core GAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGAGGG(nucleotide sequence) CTCAGCTGTCTCCGTCTTTCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAATGATGTAATGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTACTCATGGTGTTTATTTCTACTCACC AATTTCCATTAATTACCGCACA109 ADX_5417_E01 w/N ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAleader (nucleotide CCCCCACCAGCCTGCTGATCAGCTGGAGGGCTCAGCTGTCTCCGTCsequence with N-terminal TTTCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTmethionine) GTCCAGGAGTTCACTGTGCCTAATGATGTAATGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTACTCATGGTGTTTATTTCTACTCACCAATTTCCATTAATTAC CGCACA 110ADX_5417_E01 w/N ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAleader and modified C- CCCCCACCAGCCTGCTGATCAGCTGGAGGGCTCAGCTGTCTCCGTCterminus including PC TTTCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT(nucleotide sequence with GTCCAGGAGTTCACTGTGCCTAATGATGTAATGACAGCTACCATCAN-terminal methionine) GCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTACTCATGGTGTTTATTTCTACTCACCAATTTCCATTAATTAC CGCACACCGTGC 111ADX_5417_E01 w/N ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCAleader and C tail + his tagCCCCCACCAGCCTGCTGATCAGCTGGAGGGCTCAGCTGTCTCCGTC (nucleotide sequence withTTTCTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCT N-terminal methionine)GTCCAGGAGTTCACTGTGCCTAATGATGTAATGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTACTCATGGTGTTTATTTCTACTCACCAATTTCCATTAATTACCGCACACCGTGCCACCATCACCACCACCACTGA 112 ATI_1420_A10 coreEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQEFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRT 113 ATI_1420_A10 BC loop PYPSYYIE 114ATI_1420_A10 DE loop IRHPGMLEFG 115 ATI_1420_A10 FG loop VTDGASIASYAFP116 ATI_1420_A10 w/N GVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQleader EFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRT 117 ATI_1420_A10 w/NGVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQ leader + his tagEFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRTHHH HHH 118 ATI_1420_A10 w/NGVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQ leader and C tailEFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRTEID KPSQ 119ATI_1420_A10 w/N leader GVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQand C tail + his tag EFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRTEIDKPSQHHHHHH 120 ATI_1420_A10 w/N leaderGVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQ and modified C-terminusEFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRTPC including PC 121ATI_1420_A10 w/N GVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPVQleader and modified C- EFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRTPCHterminus including PC + HHHHH his tag 122 ATI_1420_A10-fullMGVSDVPRDLEVVAATPTSLLISWPYPSYYIEYRITYGETGGNSPV lengthQEFTVQSMKATISGLKPGVDYTITVYAIRHPGMLEFGISINYRTEI DKPSQHHHHHH 123ATI_1420_B09 core EVVAATPTSLLISWHKFSSLMSYRITYGETGGNSPVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRT 124 ATI_1420_B09 BC loop HKFSSLMS 125ATI_1420_B09 DE loop GSVN 126 ATI_1420_B09 FG loop IHNVGF 127ATI_1420_B09 w/N GVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSP leaderVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRT 128 ATI_1420_B09 w/NGVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSP leader + his tagVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTHHH HHH 129 AATI_1420_B09 w/NGVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSP leader and C tailVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTEID KPSQ 130 ATI_1420_B09 w/NGVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSP leader and C tail + his tagVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTEID KPSQHHHHHH 131ATI_1420_B09 w/N GVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSPleader and modified C- VQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTPCterminus including PC 132 ATI_1420_B09 w/NGVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSP leader and modified C-VQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTPCH terminus including PC +HHHHH his tag 133 ATI_1420_B09-fullMGVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNS lengthPVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTEI DKPSQHHHHHH 134ATI_1420_C02 core EVVAATPTSLLISWRIKSYYAYRITYGETGGNSPVQEFTVRQHVATISGLKPGVDYTITVYARLGDVELVYEISINYRT 135 ATI_1420_C02 BC loop RIKSYYA 136ATI_1420_C02 DE loop RQHV 137 ATI_1420_C02 FG loop RLGDVELVYE 138ATI_1420_C02 w/N GVSDVPRDLEVVAATPTSLLISWRIKSYYAYRITYGETGGNSPV leaderQEFTVRQHVATISGLKPGVDYTITVYARLGDVELVYEISINYRT 139 ATI_1420_C02 w/NGVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSP leader + his tagVQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTHHH HHH 140 ATI_1420_C02 w/NGVSDVPRDLEVVAATPTSLLISWRIKSYYAYRITYGETGGNSPV leader and C tailQEFTVRQHVATISGLKPGVDYTITVYARLGDVELVYEISINYRT EIDKPSQ 141ATI_1420_C02 w/N GVSDVPRDLEVVAATPTSLLISWRIKSYYAYRITYGETGGNSPVleader and C tail + his tag QEFTVRQHVATISGLKPGVDYTITVYARLGDVELVYEISINYRTEIDKPSQHHHHHH 142 ATI_1420_C02 w/NGVSDVPRDLEVVAATPTSLLISWRIKSYYAYRITYGETGGNSPV leader and modified C-QEFTVRQHVATISGLKPGVDYTITVYARLGDVELVYEISINYRT terminus including PC PC143 ATI_1420_C02 w/N GVSDVPRDLEVVAATPTSLLISWHKFSSLMSYRITYGETGGNSPleader and modified C- VQEFTVGSVNATISGLKPGVDYTITVYAIHNVGFISINYRTPCHterminus including PC + HHHHH his tag 144 ATI_1420_C02-fullMGVSDVPRDLEVVAATPTSLLISWRIKSYYAYRITYGETGGNSP lengthVQEFTVRQHVATISGLKPGVDYTITVYARLGDVELVYEISINYR TEIDKPSQHHHHHH 145ATI_1420_C11 core EVVAATPTSLLISWMYPLKSVPYRITYGETGGNSPVQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYRT 146 ATI_1420_C11 BC loop MYPLKSVP147 ATI_1420_C11 DE loop YSSG 148 ATI_1420_C11 FG loop MSYSTYHAFM 149ATI_1420_C11 w/N leader GVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNSPVQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYR T 150ATI_1420_C11 w/N leader GVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNSP +his tag VQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYR THHHHHH 151ATI_1420_C11 w/N leader GVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNSPand C tail VQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYR TEIDKPSQ 152ATI_1420_C11 w/N leader GVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNSPand C tail + his tag VQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYRTEIDKPSQHHHHHH 153 ATI_1420_C11 w/N leaderGVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNSP and modified C-terminusVQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYR including PC TPC 154ATI_1420_C11 w/N leader GVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNSPand modified C-terminus VQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINYRincluding PC + his tag TPCHHHHHH 155 AATI_1420_C11-fullMGVSDVPRDLEVVAATPTSLLISWMYPLKSVPYRITYGETGGNS lengthPVQEFTVYSSGATISGLKPGVDYTITVYAMSYSTYHAFMISINY RTEIDKPSQHHHHHH 156ATI_1420_D01 core EVVAATPTSLLISWRTVPETDYRITYGETGGNSPVQEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRT 157 ATI_1420_D01 BC loop RTVPETD 158ATI_1420_D01 DE loop PDNT 159 ATI_1420_D01 FG loop LETAHYNRDY 160ATI_1420_D01 w/N GVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSPV leaderQEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRT 161 ATI_1420_D01 w/NGVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSPV leader + his tagQEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRT HHHHHH 162 ATI_1420_D01 w/NGVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSPV leader and C tailQEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRT EIDKPSQ 163ATI_1420_D01 w/N GVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSPVleader and C tail + his tag QEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRTEIDKPSQHHHHHH 164 ATI_1420_D01 w/NGVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSPV leader and modified C-QEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRT terminus including PC PC165 ATI_1420_D01 w/N GVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSPVleader and modified C- QEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYRTterminus including PC + PCHHHHHH his tag 166 ATI_1420_D01-fullMGVSDVPRDLEVVAATPTSLLISWRTVPETDYRITYGETGGNSP lengthVQEFTVPDNTATISGLKPGVDYTITVYALETAHYNRDYISINYR TEIDKPSQHHHHHH 167ATI_1420_D05 core EVVAATPTSLLISWTAYYSTIKYRITYGETGGNSPVQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISINYRT 168 ATI_1420_D05 BC loop TAYYSTIK169 ATI_1420_D05 DE loop GPKHH 170 ATI_1420_D05 FG loop YNTKPGYHAHQ 171ATI_1420_D05 w/N GVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNSP leaderVQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISIN YRT 172 ATI_1420_D05 w/NGVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNSP leader + his tagVQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISIN YRTHHHHHH 173ATI_1420_D05 w/N GVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNSPleader and C tail VQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISINYRTEIDKPSQ 174 ATI_1420_D05 w/NGVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNSP leader and C tail + his tagVQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISIN YRTEIDKPSQHHHHHH 175ATI_1420_D05 w/N GVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNSPleader and modified C- VQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISINterminus including PC YRTPC 176 ATI_1420_D05 w/NGVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNSP leader and modified C-VQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISIN terminus including PC +YRTPCHHHHHH his tag 177 ATI_1420_D05-fullMGVSDVPRDLEVVAATPTSLLISWTAYYSTIKYRITYGETGGNS lengthPVQEFTVGPKHHATISGLKPGVDYTITVYAYNTKPGYHAHQISI NYRTEIDKPSQHHHHHH 178ATI_1420_D10 core EVVAATPTSLLISWRIPSYHIQYRITYGETGGNSPVQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYRT 179 ATI_1420_D10 BC loop RIPSYHIQ180 ATI_1420_D10 DE loop YQKY 181 ATI_1420_D10 FG loop VSPPKQLRFG 182ATI_1420_D10 w/N GVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNSP leaderVQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYR T 183 ATI_1420_D10 w/NGVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNSP leader + his tagVQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYR THHHHHH 184ATI_1420_D10 w/N GVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNSPleader and C tail VQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYR TEIDKPSQ185 ATI_1420_D10 w/N GVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNSPleader and C tail + his tag VQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYRTEIDKPSQHHHHHH 186 ATI_1420_D10 w/NGVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNSP leader and modified C-VQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYR terminus including PC TPC187 ATI_1420_D10 w/N GVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNSPleader and modified C- VQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINYRterminus including PC + TPCHHHHHH his tag 188 ATI_1420_D010-fullMGVSDVPRDLEVVAATPTSLLISWRIPSYHIQYRITYGETGGNS lengthPVQEFTVYQKYATISGLKPGVDYTITVYAVSPPKQLRFGISINY RTEIDKPSQHHHHHH 189ATI_1420_F10_core EVVAATPTSLLISWPAPPSYVFYRITYGETGGNSPVQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRT 190 ATI_1420_F10 BC loop PAPPSYVF 191ATI_1420_F10 DE loop YPYM 192 ATI_1420_F10 FG loop YTSGFS 193ATI_1420_F10 w/N leader GVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSPVQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRT 194 ATI_1420_F10 w/NGVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSP leader + his tagVQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRTHHH HHH 195ATI_1420_F10 w/N leader GVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSPand C tail VQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRTEID KPSQ 196ATI_1420_F10 w/N GVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSPleader and C tail + his tag VQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRTEIDKPSQHHHHHH 197 ATI_1420_F10 w/NGVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSP leader and modified C-VQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRTPC terminus including PC 198ATI_1420_F10 w/N GVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSPleader and modified C- VQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRTPCHterminus including PC + HHHHH his tag 199 ATI_1420_F10-full lengthMGVSDVPRDLEVVAATPTSLLISWPAPPSYVFYRITYGETGGNSPVQEFTVYPYMATISGLKPGVDYTITVYAYTSGFSISINYRTEI DKPSQHHHHHH 200ATI_1421_C05_core EVVAATPTSLLISWYMDHKSKYRITYGETGGNSPVQEFTVPDQRATSGLKPGVDYTITVYALSEAHYLRDKISINYRT 201 ATI_1421_C05 BC loop YMDHKSK 202ATI_1421_C05 DE loop PDQR 203 ATI_1421_C05 FG loop LSEAHYLRDK 204ATI_1421_C05 w/N GVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSPV leaderQEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYRT 205 ATI_1421_C05 w/NGVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSPV leader + his tagQEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYRT HHHHHH 206 ATI_1421_C05 w/NGVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSPV leader and C tailQEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYRT EIDKPSQ 207ATI_1421_C05 w/N GVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYRTEIDKPSQHHHHHH 208 ATI_1421_C05 w/NGVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSPV leader and modified C-QEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYRT terminus including PC PC209 ATI_1421_C05 w/N GVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSPVleader and modified C- QEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYRTterminus including PC + PCHHHHHH his tag 210 ATI_1421_C05-fullMGVSDVPRDLEVVAATPTSLLISWYMDHKSKYRITYGETGGNSP lengthVQEFTVPDQRATISGLKPGVDYTITVYALSEAHYLRDKISINYR TEIDKPSQHHHHHH 211ATI_1421_C06 core EVVAATPTSLLISWENLASYQYRITYGETGGNSPVQEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRT 212 ATI_1421_C06 BC loop ENLASYQ 213ATI_1421_C06 DE loop PDQA 214 ATI_1421_C06 FG loop LQTAHYYRQH 215ATI_1421_C06 w/N GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leaderQEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRT 216 ATI_1421_C06 w/NGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leader + his tagQEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRT HHHHHH 217ATI_1421_C06 w/N leader GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPVand C tail QEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRT EIDKPSQ 218ATI_1421_C06 w/N GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRTEIDKPSQHHHHHH 219 ATI_1421_C06 w/NGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leader and modified C-QEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRT terminus including PC PC220 ATI_1421_C06 w/N GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPVleader and modified C- QEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYRTterminus including PC + PCHHHHHH his tag 221 ATI_1421_C06-fullMGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSP lengthVQEFTVPDQAATISGLKPGVDYTITVYALQTAHYYRQHISINYR TEIDKPSQHHHHHH 222ATI_1421_D05 core EVVAATPTSLLISWYYVQYNDYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 223 ATI_1421_D05 BC loop YYVQYND 224ATI_1421_D05 DE loop PDQS 225 ATI_1421_D05 FG loop LEKAHYYRQN 226ATI_1421_D05 w/N leader GVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 227 ATI_1421_D05 w/N leaderGVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSPV + his tagQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT HHHHHH 228ATI_1421_D05 w/N leader GVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSPVand C tail QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT EIDKPSQ 229ATI_1421_D05 w/N leader GVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSPVand C tail + his tag QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTEIDKPSQHHHHHH 230 ATI 1421_D05 w/N leaderGVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSPV and modified C-terminusQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT including PC PC 231ATI 1421_D05 w/N leader GVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSPVand modified C-terminus QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTincluding PC + his tag PCHHHHHH 232 ATI 1421_D05-fullMGVSDVPRDLEVVAATPTSLLISWYYVQYNDYRITYGETGGNSP lengthVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYR TEIDKPSQHHHHHH 233ATI_1421_D06 core EVVAATPTSLLISWGHNYDDEYRITYGETGGNSPVQEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRT 234 ATI_1421_D06 BC loop GHNYDDE 235ATI_1421_D06 DE loop PDQY 236 ATI_1421_D06 FG loop LAEAHVRKNH 237ATI_1421_D06 w/N GVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSPV leaderQEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRT 238 ATI_1421_D06 w/NGVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSPV leader + his tagQEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRT HHHHHH 239 ATI_1421_D06 w/NGVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSPV leader and C tailQEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRT EIDKPSQ 240ATI_1421_D06 w/N GVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRTEIDKPSQHHHHHH 241 ATI_1421_D06 w/NGVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSPV leader and modified C-QEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRT terminus including PC PC242 ATI_1421_D06 w/N GVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSPVleader and modified C- QEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYRTterminus including PC + PCHHHHHH his tag 243 ATI_1421_D06-fullMGVSDVPRDLEVVAATPTSLLISWGHNYDDEYRITYGETGGNSP lengthVQEFTVPDQYATISGLKPGVDYTITVYALAEAHVRKNHISINYR TEIDKPSQHHHHHH 244ATI_1421_E03 core EVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT 245 ATI_1421_E03 BC loop VYHYDAQ 246ATI_1421_E03 DE loop PDQK 247 ATI_1421_E03 FG loop LSEAHHKRDS 248ATI_1421_E03 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT HHHHHH 249ATI_1421_E03 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV +his tag QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT EIDKPSQ 250ATI_1421_E03 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVand C tail QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT EIDKPSQHHHHHH251 ATI_1421_E03 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVand C tail + his tag QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT PC 252ATI_1421_E03 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVand modified C-terminus QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRTincluding PC PCHHHHHH 253 ATI_1421_E03 w/N leaderGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV and modified C-terminusQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT including PC + his tag 254ATI_1421_E03-full MGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSP lengthVQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYR TEIDKPSQHHHHHH 255ATI_1421_E04 core EVVAATPTSLLISWSYNGPIEYRITYGETGGNSPVQEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRT 256 ATI_1421_E04 BC loop SYNGPIE 257ATI_1421_E04 DE loop PDQQ 258 ATI_1421_E04 FG loop LEEAHYSRQS 259ATI_1421_E04 w/N leader GVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPVQEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRT 260 ATI_1421_E04 w/N leaderGVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPV + his tagQEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRT HHHHHH 261 ATI_1421_E04 w/NGVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPV leader and C tailQEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRT EIDKPSQ 262ATI_1421_E04 w/N leader GVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPVand C tail + his tag QEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRTEIDKPSQHHHHHH 263 ATI_1421_E04 w/N leaderGVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPV and modified C-terminusQEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRT including PC PC 264ATI_1421_E04 w/N leader GVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPVand modified C-terminus QEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYRTincluding PC + his tag PCHHHHHH 265 ATI_1421_E04-full lengthMGVSDVPRDLEVVAATPTSLLISWSYNGPIEYRITYGETGGNSPVQEFTVPDQQATISGLKPGVDYTITVYALEEAHYSRQSISINYR TEIDKPSQHHHHHH 266ATI_1421_F03 core EVVAATPTSLLISWISVQTYDYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 267 ATI_1421_F03 BC loop ISVQTYD 268ATI_1421_F03 DE loop PDQS 269 ATI_1421_F03 FG loop LEKAHYYRQN 270ATI_1421_F03 w/N leader GVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 271 ATI_1421_F03 w/N leaderGVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPV + his tagQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT HHHHHH 272 ATI_1421_F03 w/NGVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPV leader and C tailQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT EIDKPSQ 273ATI_1421_F03 w/N leader GVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPVand C tail + his tag QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTEIDKPSQHHHHHH 274 ATI_1421_F03 w/N leaderGVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPV and modified C-terminusQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT includi ng PC PC 275ATI_1421_F03 w/N leader GVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPVand modified C-terminus QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTincluding PC + his tag PCHHHHHH 276 ATI_1421_F03-full lengthMGVSDVPRDLEVVAATPTSLLISWISVQTYDYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYR TEIDKPSQHHHHHH 277ATI_1421_F05 core EVVAATPTSLLISWLARHDARYRITYGETGGNSPVQEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRT 278 ATI_1421_F05 BC loop LARHDAR 279ATI_1421_F05 DE loop PDRM 280 ATI_1421_F05 FG loop LEQAHYYRLY 281ATI_1421_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPVQEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRT 282 ATI_1421_F05 w/N leaderGVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPV + his tagQEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRT HHHHHH 283ATI_1421_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPVand C tail QEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRT EIDKPSQ 284ATI_1421_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPVand C tail + his tag QEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRTEIDKPSQHHHHHH 285 ATI_1421_F05 w/N leaderGVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPV and modified C-terminusQEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRT including PC PC 286ATI_1421_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPVand modified C-terminus QEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYRTincluding PC + his tag PCHHHHHH 287 ATI_1421_F05-full lengthMGVSDVPRDLEVVAATPTSLLISWLARHDARYRITYGETGGNSPVQEFTVPDRMATISGLKPGVDYTITVYALEQAHYYRLYISINYR TEIDKPSQHHHHHH 288ATI_1421_G07 core EVVAATPTSLLISWHSPTSGITYRITYGETGGNSPVQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISINYRT 289 ATI_1421_G07 BC loop HSPTSGIT290 ATI_1421_G07 DE loop PYDPS 291 ATI_1421_G07 FG loop PYGSQYYPGYH 292ATI_1421_G07 w/N GVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNSP leaderVQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISIN YRT 293 ATI_1421_G07 w/NGVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNSP leader + his tagVQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISIN YRTHHHHHH 294ATI_1421_G07 w/N GVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNSPleader and C tail VQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISINYRTEIDKPSQ 295 ATI_1421_G07 w/NGVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNSP leader and C tail + his tagVQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISIN YRTEIDKPSQHHHHHH 296ATI_1421_G07 w/N GVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNSPleader and modified C- VQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISINterminus including PC YRTPC 297 ATI_1421_G07 w/NGVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNSP leader and modified C-VQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISIN terminus including PC +YRTPCHHHHHH his tag 298 ATI_1421_G07-fullMGVSDVPRDLEVVAATPTSLLISWHSPTSGITYRITYGETGGNS lengthPVQEFTVPYDPSATISGLKPGVDYTITVYAPYGSQYYPGYHISI NYRTEIDKPSQHHHHHH 299ATI_1421_H03 core EVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVQEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRT 300 ATI_1421_H03 BC loop VYHYDAQ 301ATI_1421_H03 DE loop PDSS 302 ATI_1421_H03 FG loop LEQAHIDRTT 303ATI_1421_H03 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV leaderQEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRT 304 ATI_1421_H03 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV leader + his tagQEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRT HHHHHH 305 ATI_1421_H03 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV leader and C tailQEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRT EIDKPSQ 306ATI_1421_H03 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVleader and C tail + his tag QEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRTEIDKPSQHHHHHH 307 ATI_1421_H03 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV leader and modified C-QEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRT terminus including PC PC308 ATI_1421_H03 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVleader and modified C- QEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYRTterminus including PC + PCHHHHHH his tag 309 ATI_1421_H03-fullMGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSP lengthVQEFTVPDSSATISGLKPGVDYTITVYALEQAHIDRTTISINYR TEIDKPSQHHHHHH 310ATI_1421_H05 core EVVAATPTSLLISWTSVLLKDYRITYGETGGNSPVQEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRT 311 ATI_1421_H05 BC loop TSVLLKD 312ATI_1421_H05 DE loop PDQH 313 ATI_1421_H05 FG loop LQNAHHERLY 314ATI_1421_H05 w/N GVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSPV leaderQEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRT 315 ATI_1421_H05 w/NGVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSPV leader + his tagQEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRT HHHHHH 316 ATI_1421_H05 w/NGVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSPV leader and C tailQEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRT EIDKPSQ 317ATI_1421_H05 w/N GVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRTEIDKPSQHHHHHH 318 ATI_1421_H05 w/NGVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSPV leader and modified C-QEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRT terminus including PC PC319 ATI_1421_H05 w/N GVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSPVleader and modified C- QEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYRTterminus including PC + PCHHHHHH his tag 320 ATI_1421_H05-fullMGVSDVPRDLEVVAATPTSLLISWTSVLLKDYRITYGETGGNSP lengthVQEFTVPDQHATISGLKPGVDYTITVYALQNAHHERLYISINYR TEIDKPSQHHHHHH 321ATI_1422_E06 core EVVAATPTSLLISWLPSYYITYRITYGETGGNSPVQEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRT 322 ATI_1422_E06 BC loop LPSYYIT 323ATI_1422_E06 DE loop SKDL 324 ATI_1422_E06 FG loop FNGSSYYTFG 325ATI_1422_E06 w/N leader GVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPVQEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRT 326 ATI_1422_E06 w/N leaderGVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPV + his tagQEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRT EIDKPSQHHHHHH 327ATI_1422_E06 w/N leader GVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPVand C tail QEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRT EIDKPSQHHHHHH328 ATI_1422_E06 w/N leader GVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPVand C tail + his tag QEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRT PC 329ATI_1422_E06 w/N leader GVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPVand modified C-terminus QEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRTincluding PC PCHHHHHH 330 ATI_1422_E06 w/N leaderGVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPV and modified C-terminusQEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYRT including PC + his tagEIDKPSQHHHHHH 331 ATI_1422_E06-full lengthMGVSDVPRDLEVVAATPTSLLISWLPSYYITYRITYGETGGNSPVQEFTVSKDLATISGLKPGVDYTITVYAFNGSSYYTFGISINYR T 332 ATI_1422_F04 coreEVVAATPTSLLISWSIPSYFISYRITYGETGGNSPVQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRT 333 ATI_1422_F04 BC loop SIPSYFIS 334ATI_1422_F04 DE loop YKNY 335 ATI_1422_F04 FG loop SEGIMFYN 336ATI_1422_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSPVQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRT 337 ATI_1422_F04 w/N leaderGVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSP + his tagVQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRTH HHHHH 338 ATI_1422_F04 w/NGVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSP leader and C tailVQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRTE IDKPSQ 339ATI_1422_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSPand C tail + his tag VQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRTEIDKPSQHHHHHH 340 ATI_1422_F04 w/N leaderGVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSP and modified C-terminusVQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRTP includingPCc 341ATI_1422_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSPand modified C-terminus VQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRTPincluding PC + his tag CHHHHHH 342 ATI_1422_F04-full lengthMGVSDVPRDLEVVAATPTSLLISWSIPSYFISYRITYGETGGNSPVQEFTVYKNYATISGLKPGVDYTITVYASEGIMFYNISINYRT EIDKPSQHHHHHH 343ATI_1422_FO5 core EVVAATPTSLLISWPYPRGPYVFYRITYGETGGNSPVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRT 344 ATI_1422_F05 BC loop PYPRGPYVF 345ATI_1422_F05 DE loop YPGQ 346 ATI_1422_F05 FG loop YTSGYV 347ATI_1422_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNSPVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRT 348 ATI_1422_F05 w/N leaderGVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNS + his tagPVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRTEI DKPSQHHHHHH 349ATI_1422_F05 w/N GVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNSleader and C tail PVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRTEI DKPSQ 350ATI_1422_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNSand C tail + his tag PVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRTEIDKPSQHHHHHH 351 ATI_1422_F05 w/N leaderGVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNS and modified C-terminusPVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRTPC including PC 352ATI_1422_F05 w/N leader GVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNSand modified C-terminus PVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRTPCincluding PC + his tag HHHHHH 353 ATI_1422_F05-full lengthMGVSDVPRDLEVVAATPTSLLISWPYPRGPYVFYRITYGETGGNSPVQEFTVYPGQATISGLKPGVDYTITVYAYTSGYVISINYRTE IDKPSQHHHHHH 354ATI_1422_H04 core EVVAATPTSLLISWYLPSYYVQYRITYGETGGNSPVQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYRT 355 ATI_1422_H04 BC loop YLPSYYVQ356 ATI_1422_H04 DE loop KSYN 357 ATI_1422_H04 FG loop RMGVYYLSYS 358ATI_1422_H04 w/N GVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNSP leaderVQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYR T 359 ATI_1422_H04 w/NGVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNSP leader + his tagVQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYR THHHHHH 360ATI_1422_H04 w/N GVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNSPleader and C tail VQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYR TEIDKPSQ361 ATI_1422_H04 w/N GVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNSPleader and C tail + his tag VQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYRTEIDKPSQHHHHHH 362 ATI_1422_H04 w/NGVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNSP leader and modified C-VQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYR terminus including PC TPC363 ATI_1422_H04 w/N GVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNSPleader and modified C- VQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINYRterminus including PC + TPCHHHHHH his tag 364 ATI_1422_H04-fullMGVSDVPRDLEVVAATPTSLLISWYLPSYYVQYRITYGETGGNS lengthPVQEFTVKSYNATISGLKPGVDYTITVYARMGVYYLSYSISINY RTEIDKPSQHHHHHH 365ATI_1422_H05 core EVVAATPTSLLISWQGQLSPSFYRITYGETGGNSPVQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRT 366 ATI_1422_H05 BC loop QGQLSPSF 367ATI_1422_H05 DE loop VAGM 368 ATI_1422_H05 FG loop TSDVYFYS 369ATI_1422_H05 w/N GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSP leaderVQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRT 370 ATI_1422_H05 w/NGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSP leader + his tagVQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRTH HHHHH 371 ATI_1422_H05 w/NGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSP leader and C tailVQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRTE IDKPSQ 372 ATI_1422_H05 w/NGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSP leader and C tail + his tagVQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRTE IDKPSQHHHHHH 373ATI_1422_H05 w/N GVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSPleader and modified C- VQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRTPterminus including PC C 374 ATI_1422_H05 w/NGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSP leader and modified C-VQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRTP terminus including PC +CHHHHHH his tag 375 -full lengthMGVSDVPRDLEVVAATPTSLLISWQGQLSPSFYRITYGETGGNSPVQEFTVVAGMATISGLKPGVDYTITVYATSDVYFYSISINYRT EIDKPSQHHHHHH 376ATI_1422_G05 core EVVAATPTSLLISWIAPYYSVIYRITYGETGGNSPVQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISINYRT 377 ATI_1422_G05 BC loop IAPYYSVI378 ATI_1422_G05 DE loop TGSGY 379 ATI_1422_G05 FG loop TYCASVASYAF 380ATI_1422_G05 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leaderVQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISIN YRT 381 ATI_1422_G05 w/NGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leader + his tagVQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISIN YRTHHHHHH 382ATI_1422_G05 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSPleader and C tail VQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISINYRTEIDKPSQ 383 ATI_1422_G05 w/NGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leader and C tail + his tagVQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISIN YRTEIDKPSQHHHHHH 384ATI_1422_G05 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSPleader and modified C- VQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISINterminus including PC YRTPC 385 ATI_1422_G05 w/NGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leader and modified C-VQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISIN terminus including PC +YRTPCHHHHHH his tag 386 ATI_1422_G05-fullMGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNS lengthPVQEFTVTGSGYATISGLKPGVDYTITVYATYCASVASYAFISI NYRTEIDKPSQHHHHHH 387ATI_1760_C02 core EVVAATPTSLLISWIAPYYSVIYRITYGETGGNSPVQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISINYRT 388 ATI_1760_C02 BC loop IAPYYSVI389 ATI_1760_C02 DE loop PGSAY 390 ATI_1760_C02 FG loop sSGASIAAYAF 391ATI_1760_C02 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leaderVQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISIN YRT 392 ATI_1760_C02 w/NGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leader + his tagVQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISIN YRTHHHHHH 393ATI_1760_C02 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSPleader and C tail VQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISINYRTEIDKPSQ 394 ATI_1760_C02 w/NGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leader and C tail + his tagVQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISIN YRTEIDKPSQHHHHHH 395ATI_1760_C02 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSPleader and modified C- VQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISINterminus including PC YRTPC 396 ATI_1760_C02 w/NGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNSP leader and modified C-VQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISIN terminus including PC +YRTPCHHHHHH his tag 397 ATI_1760_C02-fullMGVSDVPRDLEVVAATPTSLLISWIAPYYSVIYRITYGETGGNS lengthPVQEFTVPGSAYATISGLKPGVDYTITVYASSGASIAAYAFISI NYRTEIDKPSQHHHHHH 398ATI_1760_E01 core EVVAATPTSLLISWIAPYYSVKYRITYGETGGNSPVQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISINYRT 399 ATI_1760_E01 BC loop IAPYYSVK400 ATI_1760_E01 DE loop AGADY 401 ATI_1760_E01 FG loop TYGASIASYAF 402ATI_1760_E01 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSPVQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISIN YRT 403ATI_1760_E01 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSP +his tag VQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISIN YRTHHHHHH 404ATI_1760_E01 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSPleader and C tail VQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISINYRTEIDKPSQ 405 ATI_1760_E01 w/N leaderGVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSP and C tail + his tagVQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISIN YRTEIDKPSQHHHHHH 406ATI_1760_E01 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSPand modified C-terminus VQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISINincluding PC YRTPC 407 w/N leaderGVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSP and modified C-terminusVQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISIN including PC + his tagYRTPCHHHHHH 408 ATI_1760_E01-full lengthMGVSDVPRDLEVVAATPTSLLISWIAPYYSVKYRITYGETGGNSPVQEFTVAGADYATISGLKPGVDYTITVYATYGASIASYAFISI NYRTEIDKPSQHHHHHH 409ATI_1760_F01 core EVVAATPTSLLISWIAPYYAVMYRITYGETGGNSPVQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISINYRT 410 ATI_1760_F01 BC loop IAPYYAVM411 ATI_1760_F01 DE loop PGGGY 412 ATI_1760_F01 FG loop TGGASIAAYAF 413ATI_1760_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSPVQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISIN YRT 414ATI_1760_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSP +his tag VQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISIN YRTHHHHHH 415ATI_1760_F01 w/N GVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSPleader and C tail VQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISINYRTEIDKPSQ 416 ATI_1760_F01 w/N leaderGVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSP and C tail + his tagVQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISIN YRTEIDKPSQHHHHHH 417ATI_1760_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSPand modified C-terminus VQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISINincluding PC YRTPC 418 ATI_1760_F01 w/N leaderGVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSP and modified C-terminusVQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISIN including PC + his tagYRTPCHHHHHH 419 ATI_1760_F01-full lengthMGVSDVPRDLEVVAATPTSLLISWIAPYYAVMYRITYGETGGNSPVQEFTVPGGGYATISGLKPGVDYTITVYATGGASIAAYAFISI NYRTEIDKPSQHHHHHH 420ATI_1494_D03 core EVVAATPTSLLISWSYPSYHLYRITYGETGGNSPVQEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRT 421 ATI_1494_D03 BC loop SYPSYHL 422ATI_1494_D03 DE loop HIDY 423 ATI_1494_D03 FG loop QSPPYDIYYE 424ATI_1494_D03 w/N GVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSPV leaderQEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRT 425 ATI_1494_D03 w/NGVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSPV leader + his tagQEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRT HHHHHH 426 ATI_1494_D03 w/NGVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSPV leader and C tailQEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRT EIDKPSQ 427ATI_1494_D03 w/N GVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSPVleader and C tail + his tag QEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRTEIDKPSQHHHHHH 428 ATI_1494_D03 w/NGVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSPV leader and modified C-QEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRT terminus including PC PC429 ATI_1494_D03 w/N GVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSPVleader and modified C- QEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYRTterminus including PC + PCHHHHHH his tag 430 ATI_1494_D03-fullMGVSDVPRDLEVVAATPTSLLISWSYPSYHLYRITYGETGGNSP lengthVQEFTVHIDYATISGLKPGVDYTITVYAQSPPYDIYYEISINYR TEIDKPSQHHHHHH 431ATI_1494_D04 core EVVAATPTSLLISWMESSSNSYRITYGETGGNSPVQEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRT 432 ATI_1494_D04 BC loop MESSSNS 433ATI_1494_D04 DE loop PDQL 434 ATI_1494_D04 FG loop LANAHYMRVG 435ATI_1494_D04 w/N GVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSPV leaderQEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRT 436 ATI_1494_D04 w/NGVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSPV leader + his tagQEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRT HHHHHH 437 ATI_1494_D04 w/NGVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSPV leader and C tailQEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRT EIDKPSQ 438ATI_1494_D04 w/N GVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRTEIDKPSQHHHHHH 439 ATI_1494_D04 w/NGVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSPV leader and modified C-QEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRT terminus including PC PC440 ATI_1494_D04 w/N GVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSPVleader and modified C- QEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYRTterminus including PC + PCHHHHHH his tag 441 ATI_1494_D04-fullMGVSDVPRDLEVVAATPTSLLISWMESSSNSYRITYGETGGNSP lengthVQEFTVPDQLATISGLKPGVDYTITVYALANAHYMRVGISINYR TEIDKPSQHHHHHH 442ATI_1523_A08 core EVVAATPTSLLISWISVQTYXYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 443 ATI_1523_A08 BC loop ISVQTYX 444ATI_1523_A08 DE loop PDQS 445 ATI_1523_A08 FG loop LEKAHYYRQN 446ATI_1523_A08 w/N GVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSPV leaderQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 447 ATI_1523_A08 w/NGVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSPV leader + his tagQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT HHHHHH 448 ATI_1523_A08 w/NGVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSPV leader and C tailQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT EIDKPSQ 449ATI_1523_A08 w/N GVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTEIDKPSQHHHHHH 450 ATI_1523_A08 w/NGVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSPV leader and modified C-QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT terminus including PC PC451 ATI_1523_A08 w/N GVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSPVleader and modified C- QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTterminus including PC + PCHHHHHH his tag 452 ATI_1523_A08-fullMGVSDVPRDLEVVAATPTSLLISWISVQTYXYRITYGETGGNSP lengthVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYR TEIDKPSQHHHHHH 453ATI_1523_B10 core EVVAATPTSLLISWVYHYDXQYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT 454 ATI_1523_B10 BC loop VYHYDXQ 455ATI_1523_B10 DE loop PDQK 456 ATI_1523_B10 FG loop LSEAHHKRDS 457ATI_1523_B10 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSPV leaderQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT 458 ATI_1523_B10 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSPV leader + his tagQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT HHHHHH 459 ATI_1523_B10 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSPV leader and C tailQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT EIDKPSQ 460ATI_1523_B10 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRTEIDKPSQHHHHHH 461 ATI_1523_B10 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSPV leader and modified C-QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT terminus including PC PC462 ATI_1523_B10 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSPVleader and modified C- QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRTterminus including PC + PCHHHHHH his tag 463 ATI_1523_B10-fullMGVSDVPRDLEVVAATPTSLLISWVYHYDXQYRITYGETGGNSP lengthVQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYR TEIDKPSQHHHHHH 464ATI_1523_C07 core EVVAATPTSLLISWRMHTDPDYRITYGETGGNSPVQEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRT 465 ATI_1523_C07 BC loop RMHTDPD 466ATI_1523_C07 DE loop PDQE 467 ATI_1523_C07 FG loop IQTAHYYRIN 468ATI_1523_C07 w/N GVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSPV leaderQEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRT 469 ATI_1523_C07 w/NGVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSPV leader + his tagQEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRT HHHHHH 470 ATI_1523_C07 w/NGVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSPV leader and C tailQEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRT EIDKPSQ 471ATI_1523_C07 w/N GVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRTEIDKPSQHHHHHH 472 ATI_1523_C07 w/NGVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSPV leader and modified C-QEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRT terminus including PC PC473 ATI_1523_C07 w/N GVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSPVleader and modified C- QEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYRTterminus including PC + PCHHHHHH his tag 474 ATI_1523_C07-fullMGVSDVPRDLEVVAATPTSLLISWRMHTDPDYRITYGETGGNSP lengthVQEFTVPDQEATISGLKPGVDYTITVYAIQTAHYYRINISINYR TEIDKPSQHHHHHH 475ATI_1523_D07 core EVVAATPTSLLISWENLASYQYRITYGETGGNSPVQEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRT 476 ATI_1523_D07 BC loop ENLASYQ 477ATI_1523_D07 DE loop PDVQ 478 ATI_1523_D07 FG loop LPYIHMKQRV 479ATI_1523_D07 w/N GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leaderQEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRT 480 ATI_1523_D07 w/NGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leader + his tagQEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRT HHHHHH 481 ATI_1523_D07 w/NGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leader and C tailQEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRT EIDKPSQ 482ATI_1523_D07 w/N GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPVleader and C tail + his tag QEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRTEIDKPSQHHHHHH 483 ATI_1523_D07 w/NGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPV leader and modified C-QEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRT terminus including PC PC484 ATI_1523_D07 w/N GVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSPVleader and modified C- QEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYRTterminus including PC + PCHHHHHH his tag 485 ATI_1523_D07-fullMGVSDVPRDLEVVAATPTSLLISWENLASYQYRITYGETGGNSP lengthVQEFTVPDVQATISGLKPGVDYTITVYALPYIHMKQRVISINYR TEIDKPSQHHHHHH 486ATI_1523_D08 core EVVAATPTSLLISWMRYYDAYYRITYGETGGNSPVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 487 ATI_1523_D08 BC loop MRYYDAY 488ATI_1523_D08 DE loop PDQS 489 ATI_1523_D08 FG loop LEKAHYYRQN 490ATI_1523_D08 w/N GVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSPV leaderQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT 491 ATI_1523_D08 w/NGVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSPV leader + his tagQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT HHHHHH 492 ATI_1523_D08 w/NGVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSPV leader and C tailQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT EIDKPSQ 493ATI_1523_D08 w/N GVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTEIDKPSQHHHHHH 494 ATI_1523_D08 w/NGVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSPV leader and modified C-QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRT terminus including PC PC495 ATI_1523_D08 w/N GVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSPVleader and modified C- QEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYRTterminus including PC + PCHHHHHH his tag 496 ATI_1523_D08-fullMGVSDVPRDLEVVAATPTSLLISWMRYYDAYYRITYGETGGNSP lengthVQEFTVPDQSATISGLKPGVDYTITVYALEKAHYYRQNISINYR TEIDKPSQHHHHHH 497ATI_1523_E08 core EVVAATPTSLLISWHHYQHYEYRITYGETGGNSPVQEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRT 498 ATI_1523_E08 BC loop HHYQHYE 499ATI_1523_E08 DE loop PDMG 500 ATI_1523_E08 FG loop LEEAHSDRSS 501ATI_1523_E08 w/N leader GVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPVQEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRT 502 ATI_1523_E08 w/N leaderGVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPV + his tagQEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRT HHHHHH 503ATI_1523_E08 w/N leader GVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPVand C tail QEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRT EIDKPSQ 504ATI_1523_E08 w/N leader GVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPVand C tail + his tag QEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRTEIDKPSQHHHHHH 505 ATI_1523_E08 w/N leaderGVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPV and modified C-terminusQEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRT including PC PC 506ATI_1523_E08 w/N leader GVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPVand modified C-terminus QEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYRTincluding PC + his tag PCHHHHHH 507 ATI_1523_E08-full lengthMGVSDVPRDLEVVAATPTSLLISWHHYQHYEYRITYGETGGNSPVQEFTVPDMGATISGLKPGVDYTITVYALEEAHSDRSSISINYR TEIDKPSQHHHHHH 508ATI_1523_F01 core EVVAATPTSLLISWYKPSTIVTYRITYGETGGNSPVQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRT 509 ATI_1523_F01 BC loop YKPSTIVT 510ATI_1523_F01 DE loop YGYN 511 ATI_1523_F01 FG loop VHGVRF 512ATI_1523_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSPVQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRT 513 ATI_1523_F01 w/N leaderGVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSP + his tagVQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRTHHH HHH 514ATI_1523_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSPand C tail VQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRTEID KPSQ 515ATI_1523_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSPand C tail + his tag VQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRTEIDKPSQHHHHHH 516 ATI_1523_F01 w/N leaderGVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSP and modified C-terminusVQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRTPC including PC 517ATI_1523_F01 w/N leader GVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSPand modified C-terminus VQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRTPCHincluding PC + his tag HHHHH 518 ATI_1523_F01-full lengthMGVSDVPRDLEVVAATPTSLLISWYKPSTIVTYRITYGETGGNSPVQEFTVYGYNATISGLKPGVDYTITVYAVHGVRFISINYRTEI DKPSQHHHHHH 519ATI_1523_F04 core EVVAATPTSLLISWGGSLSPTFYRITYGETGGNSPVQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRT 520 ATI_1523_F04 BC loop GGSLSPTF 521ATI_1523_F04 DE loop TYQG 522 ATI_1523_F04 FG loop TEGIVYYQ 523ATI_1523_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSPVQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRT 524 ATI_1523_F04 w/N leaderGVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSP + his tagVQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRTH HHHHH 525ATI_1523_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSPand C tail VQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRTE IDKPSQ 526ATI_1523_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSPand C tail + his tag VQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRTEIDKPSQHHHHHH 527 ATI_1523_F04 w/N leaderGVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSP and modified C-terminusVQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRTP including PC C 528ATI_1523_F04 w/N leader GVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSPand modified C-terminus VQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRTPincluding PC + his tag CHHHHHH 529 ATI_1523_F04-full lengthMGVSDVPRDLEVVAATPTSLLISWGGSLSPTFYRITYGETGGNSPVQEFTVTYQGATISGLKPGVDYTITVYATEGIVYYQISINYRT EIDKPSQHHHHHH 530ATI_1523_F08 core EVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRT 531 ATI_1523_F08 BC loop VYHYDAQ 532ATI_1523_F08 DE loop PDQK 533 ATI_1523_F08 FG loop LPRAHMDRSH 534ATI_1523_F08 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRT 535 ATI_1523_F08 w/N leaderGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV + his tagQEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRT HHHHHH 536ATI_1523_F08 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVand C tail QEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRT EIDKPSQ 537ATI_1523_F08 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVand C tail + his tag QEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRTEIDKPSQHHHHHH 538 ATI_1523_F08 w/N leaderGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPV and modified C-terminusQEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRT including PC PC 539ATI_1523_F08 w/N leader GVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVand modified C-terminus QEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYRTincluding PC + his tag PCHHHHHH 540 ATI_1523_F08-full lengthMGVSDVPRDLEVVAATPTSLLISWVYHYDAQYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALPRAHMDRSHISINYR TEIDKPSQHHHHHH 541ATI_1523_G06 core EVVAATPTSLLISWRIKSYHKYRITYGETGGNSPVQEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRT 542 ATI_1523_G06 BC loop RIKSYHK 543ATI_1523_G06 DE loop RSYA 544 ATI_1523_G06 FG loop IMEETHLAYA 545ATI_1523_G06 w/N GVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSPV leaderQEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRT 546 ATI_1523_G06 w/NGVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSPV leader + his tagQEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRT HHHHHH 547 ATI_1523_G06 w/NGVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSPV leader and C tailQEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRT EIDKPSQ 548ATI_1523_G06 w/N GVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSPVleader and C tail + his tag QEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRTEIDKPSQHHHHHH 549 ATI_1523_G06 w/NGVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSPV leader and modified C-QEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRT terminus including PC PC550 ATI_1523_G06 w/N GVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSPVleader and modified C- QEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYRTterminus including PC + PCHHHHHH his tag 551 ATI_1523_G06-fullMGVSDVPRDLEVVAATPTSLLISWRIKSYHKYRITYGETGGNSP lengthVQEFTVRSYAATISGLKPGVDYTITVYAIMEETHLAYAISINYR TEIDKPSQHHHHHH 552ATI_1523_G07 core EVVAATPTSLLISWVYPQADDYRITYGETGGNSPVQEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRT 553 ATI_1523_G07 BC loop VYPQADD 554ATI_1523_G07 DE loop PDQN 555 ATI_1523_G07 FG loop LAEAHLVRIY 556ATI_1523_G07 w/N GVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSPV leaderQEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRT 557 ATI_1523_G07 w/NGVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSPV leader + his tagQEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRT HHHHHH 558 ATI_1523_G07 w/NGVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSPV leader and C tailQEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRT EIDKPSQ 559ATI_1523_G07 w/N GVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRTEIDKPSQHHHHHH 560 ATI_1523_G07 w/NGVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSPV leader and modified C-QEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRT terminus including PC PC561 ATI_1523_G07 w/N GVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSPVleader and modified C- QEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYRTterminus including PC + PCHHHHHH his tag 562 ATI_1523_G07-fullMGVSDVPRDLEVVAATPTSLLISWVYPQADDYRITYGETGGNSP lengthVQEFTVPDQNATISGLKPGVDYTITVYALAEAHLVRIYISINYR TEIDKPSQHHHHHH 563ATI_1523_H07 core EVVAATPTSLLISWVYHYDAXYRITYGETGGNSPVQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT 564 ATI_1523_H07 BC loop VYHYDAX 565ATI_1523_H07 DE loop PDQK 566 ATI_1523_H07 FG loop LSEAHHKRDS 567ATI_1523_H07 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPV leaderQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT 568 ATI_1523_H07 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPV leader + his tagQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT HHHHHH 569 ATI_1523_H07 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPV leader and C tailQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT EIDKPSQ 570ATI_1523_H07 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPVleader and C tail + his tag QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRTEIDKPSQHHHHHH 571 ATI_1523_H07 w/NGVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPV leader and modified C-QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT terminus including PC PC572 ATI_1523_H07 w/N GVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPVleader and modified C- QEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRTterminus including PC + PCHHHHHH his tag 573 ATI_1523_H07-fullGVSDVPRDLEVVAATPTSLLISWVYHYDAXYRITYGETGGNSPV lengthQEFTVPDQKATISGLKPGVDYTITVYALSEAHHKRDSISINYRT EIDKPSQHHHHHH 574N-terminal leader MGVSDVPRDL 575 N-terminal leader GVSDVPRDL 576N-terminal leader XnSDVPRDL 577 N-terminal leader XnDVPRDL 578N-terminal leader XnVPRDL 579 N-terminal leader XnPRDL 580N-terminal leader XnRDL 581 N-terminal leader XnDL 582 N-terminal leaderMASTSG 583 N-terminal leader METDTLLLWVLLLWVPGSTG 584 C-terminal tailEIEK 585 C-terminal tail EGSGC 586 C-terminal tail EIEKPCQ 587C-terminal tail EIEKPSQ 588 C-terminal tail EIEKP 589 C-terminal tailEIEKPS 590 C-terminal tail EIEKPC 591 C-terminal tail EIDK 592C-terminal tail EIDKPCQ 593 C-terminal tail EIDKPSQ 594 C-terminal tailEIEPKSS 595 C-terminal tail EIDKPC 596 C-terminal tail EIDKP 597C-terminal tail EIDKPS 598 C-terminal tail EIDKPSQLE 599 C-terminal tailEIEDEDEDEDED 600 C-terminal tail EGSGS 601 C-terminal tail EIDKPCQLE 602C-terminal tail EIDKPSQHHHHHH 603 C-terminal tail GSGCHHHHHH 604C-terminal tail EGSGCHHHHHH 605 C-terminal tail PIDK 606 C-terminal tailPIEK 607 C-terminal tail PIDKP 608 C-terminal tail PIEKP 609C-terminal tail PIDKPS 610 C-terminal tail PIEKPS 611 C-terminal tailPIDKPC 612 C-terminal tail PIEKPC 613 C-terminal tail PIDKPSQ 614C-terminal tail PIEKPSQ 615 C-terminal tail PIDKPCQ 616 C-terminal tailPIEKPCQ 617 C-terminal tail PHHHHHH 618 C-terminal tail PCHHHHHH 6196X-His tag HHHHHH 620 Human IgG1 Fc domainDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 621 Core hinge region of FcDKTHTCPPCPAPELLG 622 Exemplary hinge sequence EPKSSDKTHTCPPCPAPELLGGPS623 Exemplary hinge sequence EPKSSDKTHTCPPCPAPELLGGSS 624Exemplary hinge sequence EPKSSGSTHTCPPCPAPELLGGSS 625Exemplary hinge sequence DKTHTCPPCPAPELLGGPS 626Exemplary hinge sequence DKTHTCPPCPAPELLGGSS 627 Fc with CH2 and CH3VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH regions of IgG1 forNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP Adnectin-hinge-FcIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI constructAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK628 Fc with CH2 and CH3 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHregions of IgG1 for Fc- NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPhinge-Adnectin construct IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP629 Linker 1 GAGGGGSG 630 Linker 2 EPKSSD 631 Linker 3 PVGVV 632Linker 4 ESPKAQASSVPTAQPQAEGLA 633 Linker 5 ELQLEESAAEAQDGELD 634Linker 6 GQPDEPGGS 635 Linker 7 GGSGSGSGSGSGS 636 Linker 8ELQLEESAAEAQEGELE 637 Linker 9 GSGSG 638 Linker 10 GSGC 639 Linker 11AGGGGSG 640 Linker 12 GSGS 641 Linker 13 QPDEPGGS 642 Linker 14 GSGSGS643 Linker 15 TVAAPS 644 Linker 16 KAGGGGSG 645 Linker 17 KGSGSGSGSGSGS646 Linker 18 KQPDEPGGS 647 Linker 19 KELQLEESAAEAQDGELD 648 Linker 20KTVAAPS 649 Linker 21 KAGGGGSGG 650 Linker 22 KGSGSGSGSGSGSG 651Linker 23 KQPDEPGGSG 652 Linker 24 KELQLEESAAEAQDGELDG 653 Linker 25KTVAAPSG 654 Linker 26 AGGGGSGG 655 Linker 27 AGGGGSG 656 Linker 28GSGSGSGSGSGSG 657 Linker 29 QPDEPGGSG 658 Linker 30 TVAAPSG 659Linker 31 PSTSTST 660 Linker 32 EIDKPSQ 661 Linker 33 GSGSGSGS 662Linker 34 GSGSGSGSGS 663 Linker 35 GSGSGSGSGSGS 664 Linker 36GSGSGSGSGSGSGS 665 Linker 37 GGSGSGSGSGSGS 666 Linker 38GGSGSGSGSGSGSGSG 667 Linker 39 GSEGSEGSEGSEGSE 668 Linker 40 GGSEGGSE669 Linker 41 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 670 Linker 42GGGGSGGGGSGGGGSGGGGSGGGGS 671 Linker 43 GGGGSGGGGSGGGGSG 672 Linker 44GPGPGPG 673 Linker 45 GPGPGPGPGPG 674 Linker 46 PAPAPA 675 Linker 47PAPAPAPAPAPA 676 Linker 48 PAPAPAPAPAPAPAPAPA 677 Linker 49GSGSGSGSGSGSGSGSGSGS 678 Linker 50 GGGGSGGGGSGGGGSGGGGS

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. A polypeptide comprising a fibronectin type III tenthdomain (¹⁰Fn3), wherein (a) the ¹⁰Fn3 domain comprises AB, BC, CD, DE,EF, and FG loops, (b) the ¹⁰Fn3 has at least one loop selected from loopBC, DE, and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO:1), and (c) the polypeptide specifically binds to PD-L1.
 2. Thepolypeptide of claim 1, wherein the polypeptide binds to PD-L1 with aK_(D) of 500 mM or less.
 3. The polypeptide of claim 2, wherein thepolypeptide binds to PD-L1 with a K_(D) of 100 mM or less.
 4. Thepolypeptide of any one of claims 1-3, wherein the BC, DE, and FG loopscomprise the amino acid sequences of: (a) SEQ ID NOs: 6, 7, and 8,respectively; (b) SEQ ID NOs: 21, 22, and 23, respectively; (c) SEQ IDNOs: 36, 37, and 38, respectively; (d) SEQ ID NOs: 51, 52, and 53,respectively; (e) SEQ ID NOs: 66, 67, and 68, respectively; (f) SEQ IDNOs: 81, 82, and 83, respectively; or (g) SEQ ID NOs: 97, 98, and 99,respectively.
 5. The polypeptide of any one of claims 1-4, wherein thepolypeptide comprises an amino acid sequence at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO: 5, 20, 35, 50, 65, 80, or96.
 6. The polypeptide of claim 5, wherein the polypeptide comprises anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:
 80. 7. The polypeptide of claim 5, wherein thepolypeptide comprises an amino acid sequence at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO:
 96. 8. The polypeptide ofany one of claims 1-7, wherein the polypeptide comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to thenon-BC, DE, and FG loop regions of SEQ ID NO: 5, 20, 35, 50, 65, 80, or96.
 9. The polypeptide of any one of claims 1-7, wherein the polypeptidecomprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to an amino acid sequence selected from the groupconsisting of: SEQ ID NOs: 9-15, 24-30, 39-45, 54-60, 69-75, 84-91, and100-107.
 10. The polypeptide of any one of claims 1-7 and 9, wherein thepolypeptide comprises an amino acid sequence at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop regionsof an amino acid sequence selected from the group consisting of: SEQ IDNOs: 9-15, 24-30, 39-45, 54-60, 69-75, 84-91, and 100-107.
 11. Thepolypeptide of any one of claims 1-10, wherein the polypeptide comprisesan N-terminal leader selected from the group consisting of SEQ ID NOs:112-121, and/or a C-terminal tail selected from the group consisting ofSEQ ID NOs: 122-156.
 12. The polypeptide of any one of claims 1-11,wherein the polypeptide comprises one or more pharmacokinetic (PK)moieties selected from the group consisting of polyethylene glycol,sialic acid, Fc, Fc fragment, transferrin, serum albumin, a serumalbumin binding protein, and a serum immunoglobulin binding protein. 13.The polypeptide of claim 12, wherein the PK moiety and the polypeptideare linked via at least one disulfide bond, a peptide bond, apolypeptide, a polymeric sugar or a polyethylene glycol moiety.
 14. Thepolypeptide of claim 13, wherein the PK moiety and the polypeptide arelinked via a linker with an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 167-216.
 15. A nucleic acid encoding thepolypeptide of any one of claims 1-14.
 16. The nucleic acid of claim 15,wherein the nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 16-19, 31-34, 46-49, 61-64, 76-79,92-95, and 108-111.
 17. A vector comprising the nucleic acid of claim15.
 18. A cell comprising the nucleic acid of claim
 15. 19. Acomposition comprising the polypeptide of any one of claims 1-14, and acarrier.
 20. An imaging agent comprising the polypeptide of any one ofclaims 1-14, and a detectable label.
 21. The imaging agent of claim 20,wherein the detectable label is detectable by positron emissiontomography.
 22. The imaging agent of claim 20 or 21, wherein thepolypeptide is conjugated to the detectable label by an moiety selectedfrom the group consisting of DFO, DOTA, CB-DO2A, 3p-C-DEPA, TCMC, DBCO,DIBO, BARAC, DIMAC, Oxo-DO3A, TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P,MM-TE2A, DM-TE2A, diamsar, NODASA, NODAGA, NOTA, NETA, TACN-TM, DTPA,1B4M-DTPA, CHX-A″-DTPA, TRAP, NOPO, AAZTA, DATA, H₂dedpa, H₄octapa,H₂azapa, H₅decapa, H₆phospa, HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA,EDTA, TETA, and TRITA.
 23. The imaging agent of claim 22, wherein theconjugating moiety is NODAGA.
 24. The imaging agent of any one of claims20-23, wherein the detectable label is a radionuclide.
 25. The imagingagent of claim 24, wherein the radionuclide is selected from the groupconsisting of: ⁶⁴Cu, ¹²⁴I, ^(76/77)Br, ⁸⁶Y, ⁸⁹Zr, ⁶⁸Ga, ¹⁸F, ¹¹C, ¹²⁵I,¹²⁴I, ¹³¹I, ¹²³I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁶⁸Ga, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br,⁷⁷Br, ⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, or ¹⁵³Sm.
 26. Theimaging agent of claim 25, wherein the radionuclide is ¹⁸F.
 27. Theimaging agent of claim 25, wherein the radionuclide is ⁶⁴Cu.
 28. Theimaging agent of claim 24, wherein the chelating agent is NODAGA and theradionuclide is ⁶⁴Cu.
 29. The imaging agent of claim 24, wherein theimaging agent comprises the polypeptide of claim 6, the NODAGA, and theradionuclide ⁶⁴Cu.
 30. The imaging agent of claim 24, wherein theimaging agent comprises the polypeptide of claim 7, NODAGA, and theradionuclide ⁶⁴Cu.
 31. An imaging agent comprising the polypeptide ofany one of claims 1-14, a ¹⁸F-radiolabeled prosthetic group and abifunctional conjugating (BFC) moiety, wherein the imaging agent has thefollowing structure,

wherein the ¹⁸F is ortho to the N atom, x is an integer from 1 to 8, orpharmaceutically acceptable salt thereof.
 32. The imaging agent of claim31, wherein ¹⁸F-radiolabeled prosthetic group has the followingstructure,


33. The imaging agent of claims 31 or 32, wherein the [O(CH₂)₂]_(x)moiety is present in the 1-3 configuration relative to the nitrogen onthe pyridine ring.
 34. The imaging agent of claims 31 or 32, wherein the[O(CH₂)₂]_(x) moiety is present in the 1-2 configuration relative to thenitrogen on the pyridine ring.
 35. The imaging agent of claims 31 or 32,wherein the [O(CH₂)₂]_(x) moiety is present in the 1-4 configurationrelative to the nitrogen on the pyridine ring.
 36. The imaging agent ofclaim 31, wherein ¹⁸F-radiolabeled prosthetic group has the followingstructure,


37. The imaging agent of any one of the claims 31 to 36, wherein x is aninteger from 2 to
 6. 38. The imaging agent of claim 37, wherein x is aninteger from 3 to
 5. 39. The imaging agent of claim 37, wherein x is 4.40. The imaging agent of any one of claims 31-39, wherein the[O(CH₂)₂]_(x) moiety is present in the 1-3 configuration relative to thenitrogen on the pyridine ring.
 41. The imaging agent of any one of theclaims 31-40, wherein the pyridine ring comprises an additionalsubstituent which does not interfere with the fluorination reaction. 42.The imaging agent of claim 41, wherein the substituent on the pyridinering is a C₁₋₆ alkyl.
 43. The imaging agent of claim 42, wherein thesubstituent is methyl, ethyl or propyl.
 44. The imaging agent of claim31, wherein the ¹⁸F-radiolabeled prosthetic group has the structure


45. An imaging agent comprising the polypeptide of any one of claims1-14, an ¹⁸F-radiolabeled prosthetic group and a bifunctionalconjugating (BFC) moiety, wherein the imaging agent has the followingstructure

wherein “OPEG” is [O(CH₂)₂]_(x), and x is an integer from 1 to 8, or apharmaceutically acceptable salt thereof and wherein “Protein” is thepolypeptide comprised in the imaging agent of any one of claims 1-14.46. The imaging agent of claim 45, wherein x is an integer from 2 to 6,or a pharmaceutically acceptable salt thereof.
 47. The imaging agent ofclaim 45, wherein x is an integer from 3 to 5, or a pharmaceuticallyacceptable salt thereof.
 48. The imaging agent of claim 45, wherein x is4, or a pharmaceutically acceptable salt thereof.
 49. The imaging agentof any one of claims 31 to 48, wherein the BFC is a cyclooctynecomprising a reactive group that forms a covalent bond with an amine,carboxyl, carbonyl or thiol functional group on the protein.
 50. Theimaging agent of of claim 49, wherein the cyclooctyne is selected fromthe group consisting of dibenzocyclooctyne (DIBO),biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC) anddibenzocyclooctyne (DBCO).
 51. The imaging agent of claim 50, whereinthe cyclooctyne is DBCO.
 52. The imaging agent of any one of claims 31to 51, wherein the BFC further comprises a polyethylene glycol (PEG)_(y)spacer arm, wherein y is an integer from 1 to
 8. 53. The imaging agentof claim 52, wherein y is an integer from 2 to
 6. 54. The imaging agentof claim 52, wherein y is 4 or
 5. 55. The imaging agent of claim 52,wherein the BFC is DBCO-PEG4-NHS-Ester, DBCO-Sulfo-NHS-Ester,DBCO-PEG4-Acid, DBCO-PEG4-Amine or DBCO-PEG4-Maleimide.
 56. The imagingagent of claim 55, wherein the BFC is DBCO-PEG4-Maleimide.
 57. Theimaging agent of claim 31, wherein the imaging agent has the structure:

wherein X is a polypeptide comprising the amino acid sequence of any oneof SEQ ID NOs: 13, 28, 43, 58, 73, 88, and
 104. 58. The imaging agent ofclaim 57, wherein the polypeptide comprises the amino acid sequence setforth in SEQ ID NO:
 88. 59. The imaging agent of claim 57, wherein thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:104.
 60. A kit comprising the polypeptide, composition, or imaging agentof any one of claims 1-14 and 19-59, and instructions for use.
 61. Amethod of detecting PD-L1 in a sample comprising contacting the samplewith the polypeptide of any one of claims 1-14, and detecting PD-L1. 62.A method of detecting PD-L1 positive cells in a subject comprisingadministering to the subject an imaging agent of any one of claims31-59, and detecting the imaging agent, the detected imaging agentdefining the location of the PD-L1 positive cells in the subject.
 63. Amethod of detecting PD-L1-expressing tumors in a subject comprisingadministering to the subject an imaging agent of any one of claims31-59, and detecting the imaging agent, the detected imaging agentdefining the location of the tumor in the subject.
 64. The method ofclaim 62 or 63, wherein the imaging agent is detected by positronemission tomography.
 65. A method of obtaining an image of the imagingagent of any one of claims 31-59, the method comprising, a)administering the imaging agent to a subject; and b) imaging in vivo thedistribution of the imaging agent by positron emission tomography.
 66. Amethod of obtaining a quantitative image of tissues or cells expressingPD-L1, the method comprising contacting the cells or tissue with theimaging agent of any one of claims 31-59, and detecting or quantifyingthe tissue expressing PD-L1 using positron emission tomography.
 67. Amethod for detecting a PD-L1-expressing tumor comprising administeringan imaging-effective amount of the imaging agent of any one of claims31-59 to a subject having a PD-L1-expressing tumor, and detecting theradioactive emissions of said imaging agent in the tumor using positronemission tomography, wherein the radioactive emissions are detected inthe tumor.
 68. A method of diagnosing the presence of a PD-L1-expressingtumor in a subject, the method comprising (a) administering to a subjectin need thereof the imaging agent of any one of claims 31-59; and (b)obtaining an radio-image of at least a portion of the subject to detectthe presence or absence of the imaging agent; wherein the presence andlocation of the imaging agent above background is indicative of thepresence and location of the disease.
 69. A method of monitoring theprogress of an anti-tumor therapy against PD-L1-expressing tumors in asubject, the method comprising (a) administering to a subject in needthereof the imaging agent of any one of claims 31-59 at a first timepoint and obtaining an image of at least a portion of the subject todetermine the size of the tumor; (b) administering an anti-tumor therapyto the subject; (c) administering to the subject the imaging agent atone or more subsequent time points and obtaining an image of at least aportion of the subject at each time point; wherein the dimension andlocation of the tumor at each time point is indicative of the progressof the disease.